Activatable clostridial toxins

ABSTRACT

Compositions comprising activatable recombinant neurotoxins and polypeptides derived therefrom. The invention also comprises nucleic acids encoding such polypeptides, and methods of making such polypeptides and nucleic acids.

This application is a continuation-in-part and claims priority pursuantto 35 U.S.C. § 120 to U.S. patent application Ser. No. 11/326,265, filedJan. 6, 2006, a divisional application that claims priority pursuant to35 U.S.C. § 120 to U.S. patent application Ser. No. 09/648,692, filedAug. 8, 2000, an application that claims priority pursuant to pursuantto 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No.60/150,710 filed on Aug. 5, 1999; and claims priority pursuant to 35U.S.C. § 120 to U.S. patent application Ser. No. 11/776,075 filed onJul. 11, 2007, an application that claims priority pursuant to 35 U.S.C.§ 119(e) to U.S. Provisional Patent Application Ser. No. 60/807,059filed Jul. 11, 2006, each of which is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

This invention concerns methods and compositions useful in the fields ofneurobiology, molecular biology, and medicine, as well as methods forthe production of potentially toxic therapeutic agents and derivativesthereof. The invention also concerns recombinant clostridial neurotoxins(particular botulinum neurotoxins), modified versions thereof, andmethods of making such molecules, for use as therapeutic agents,transporter molecules, adducts, and the like.

BACKGROUND OF THE INVENTION

Neurotoxins, such as those obtained from Clostridium botulinum andClostridium tetani, are highly potent and specific poisons of neuralcells, and other cells when delivered within such cells for therapeuticpurposes. These Gram positive bacteria express two related but distincttoxins types, each comprising two disulfide-linked amino acid chains: alight chain (L) of about 50 KDa and a heavy chain (H) of about 100 KDa,which are wholly responsible for the symptoms of these diseases. Theholotoxin is synthesised in vivo as a single-chain, then nicked in apost-translational modification to form the active neurotoxin comprisingthe separate L and H chains.

The tetanus and botulinum toxins are among the most lethal substancesknown to man, having a lethal dose in humans of between 0.1 ng and 1 ngper kilogram of body weight. Tonello et al., Adv. Exp. Med. & Biol.389:251-260 (1996). Both toxins function by inhibiting neurotransmitterrelease in affected neurons. The tetanus neurotoxin (TeNT) acts mainlyin the central nervous system, while botulinum neurotoxin (BoNT) acts atthe neuromuscular junction and other cholinergic synapses in theperipheral nervous system; both act by inhibiting neurotransmitterrelease from the axon of the affected neuron into the synapse, resultingin paralysis.

The tetanus neurotoxin (TeNT) is known to exist in one immunologicallydistinct type; the botulinum neurotoxins (BoNT) are known to occur inseven different immunogenic types, termed BoNT/A through BoNT/G. Whileall of these types are produced by isolates of C. botulinum, two otherspecies, C. baratii and C. butyricum also produce toxins similar to /Fand /E, respectively. See e.g., Coffield et al., The Site and Mechanismof Action of Botulinum Neurotoxin in Therapy with Botulinum Toxin 3-13(Jankovic J. & Hallett M. eds. 1994), the disclosure of which isincorporated herein by reference.

Regardless of type, the molecular mechanism of intoxication appears tobe similar. In the first step of the process, the toxin binds to thepresynaptic membrane of the target neuron through a specific interactionbetween the heavy (H) chain and a cell surface receptor; the receptor isthought to be different for each type of botulinum toxin and for TeNT.Dolly et al., Seminars in Neuroscience 6:149-158 (1994), incorporated byreference herein. The carboxyl terminus of the heavy chain appears to beimportant for targeting of the toxin to the cell surface. Id.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino terminus ofthe H chain, which triggers a conformational change of the toxin inresponse to a pH of about 5.5 or lower. Endosomes are known to possess aproton pump which decreases intra endosomal pH. The conformational shiftexposes hydrophobic residues in the toxin, which permits the toxin toembed itself in the endosomal membrane. The toxin then translocatesthrough the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the H and light (L)chain. The entire toxic activity of botulinum and tetanus toxins iscontained in the L chain of the holotoxin; the L chain is a zinc (Zn⁺⁺)endopeptidase which selectively cleaves proteins essential forrecognition and docking of neurotransmitter-containing vesicles with thecytoplasmic surface of the plasma membrane, and fusion of the vesicleswith the plasma membrane. T×NT, BoNT/B BoNT/D, BoNT/F, and BoNT/G causedegradation of synaptobrevin, also called vesicle-associated membraneprotein (VAMP), a synaptosomal membrane protein. Most of the cytosolicdomain of VAMP extending from the surface of the synaptic vesicle isremoved as a result of any one of these cleavage events. Each toxin(except TeNT and BoNT/B) specifically cleaves a different bond.

BoNT/A and /E selectively cleave the plasma membrane-associated proteinSNAP-25; this protein, which is also cleaved by BoNT/C1 (Foran et al.,Biochem. 35:2630-2636 (1996)), is predominantly bound to and present onthe cytosolic surface of the plasma membrane. BoNT/C cleaves syntaxin,an integral protein having most of its mass exposed to the cytosol.Syntaxin interacts with the calcium channels at presynaptic terminalactive zones. See Tonello et al., Tetanus and Botulinum Neurotoxins inIntracellular Protein Catabolism 251-260 (Suzuki K. & Bond J. eds.1996), the disclosure of which is incorporated by reference as part ofthis specification.

Both TeNT and BoNT are taken up at the neuromuscular junction. BoNTremains within peripheral neurons, and blocks release of theneurotransmitter acetylcholine from these cells. Through its receptor,TeNT enters vesicles that move in a retrograde manner along the axon tothe soma, and is discharged into the intersynaptic space between motorneurons and the inhibitory neurons of the spinal cord. At this point,TeNT binds receptors of the inhibitory neurons, is again internalized,and the light chain enters the cytosol to block the release of theinhibitory neurotransmitters 4-aminobutyric acid (GABA) and glycine fromthese cells.

Because of its specifically localized effects, minute doses of BoNT havebeen used since 1981 as therapeutic agents in the treatment of patientssuffering from dystonias, including strabismus (misalignment of theeye), bephlarospasm (involuntary eyelid closure) and hemifacial spasm.See e.g., Borodic et al, Pharmacology and Histology of the TherapeuticApplication of Botulinum Toxin in Therapy with Botulinum Toxin 119-157(Jankovic J. & Hallett eds. 1994), hereby incorporated by referenceherein. Of the seven toxin types, BoNT/A is the most potent of theBoNTs, and the best characterized. Intramuscular injection of spastictissue with small quantities of BoNT/A has also been used effectively totreat spasticity due to brain injury, spinal cord injury, stroke,multiple sclerosis and cerebral palsy. The extent of paralysis dependson both the dose and volume delivered to the target site.

Although the L chain is the moiety responsible for neural intoxication,it must be delivered to the neural cytoplasm in order to be toxic.Similarly, the single-chain holotoxin pro-forms exhibit relatively lowtoxicity until they are cleaved at one or more peptide bonds in anexposed loop region between their H and L chains to create thefully-active mature neurotoxins. As implied in the mechanism providedabove, the H chain of each neurotoxin is essential for cell receptorbinding and endocytosis, while both the L and the H chains (and anintact disufide bond) are required for translocation of the toxin intothe cytoplasm. As indicated above, the L chain alone is responsible forthe toxicity caused by inhibition of acetylcholine secretion.

Despite the clear therapeutic efficacy of clostridial neurotoxinpreparations, industrial production of the toxin is difficult.Production of neurotoxoin from anaerobic Clostridium cultures is acumbersome and time-consuming process including a multi-steppurification protocol involving several protein precipitation steps andeither prolonged and repeated crystallisation of the toxin or severalstages of column chromatography. Significantly, the high toxicity of theproduct dictates that the procedure must be performed under strictcontainment (BL-3). During the fermentation process, the foldedsingle-chain neurotoxins are activated by endogenous clostridialproteases through a process termed nicking. This involves the removal ofapproximately 10 amino acid residues from the single-chain to create thedi-chain form in which the two chains remain covalently linked throughthe interchain disulfide bond.

The nicked neurotoxin is much more active than the unnicked form. Theamount and precise location of nicking varies with the serotypes of thebacteria producing the toxin. The differences in single-chain neurotoxinactivation and, hence, the yield of nicked toxin, are due to variationsin the type and amounts of proteolytic activity produced by a givenstrain. For example, greater than 99% of C. botulinum type Asingle-chain neurotoxin is activated by the Hall A C. botulinum strain,whereas type B and E strains produce toxins with lower amounts ofactivation (0 to 75% depending upon the fermentation time). Thus, thehigh toxicity of the mature neurotoxin plays a major part in thecommercial manufacture of neurotoxins as therapeutic agents.

The degree of activation of engineered clostridial toxins is, therefore,an important consideration for manufacture of these materials. It wouldbe a major advantage if neurotoxins such as BoNT and TeNT could beexpressed in high yield in rapidly-growing bacteria (such asheterologous E. coli cells) as relatively non-toxic single-chains (orsingle-chains having reduced toxic activity) which are safe, easy toisolate and simple to convert to the fully-active form.

With safety being a prime concern, previous work has concentrated on theexpression in E. coli and purification of individual H and L chains ofTeNT and BoNT; these isolated chains are, by themselves, non-toxic; seeLi et al., Biochemistry 33:7014-7020 (1994); Zhou et al., Biochemistry34:15175-15181 (1995), hereby incorporated by reference herein.Following the separate production of these peptide chains and understrictly controlled conditions the H and L subunits can be combined byoxidative disulphide linkage to form the neuroparalytic di-chains.Unfortunately, this strategy has several drawbacks.

Firstly, it is not practical to express and isolate large amounts of theindividual chains; in particular, in the absence of the L chain theisolated H chain is quite insoluble in aqueous solution and is highlysusceptible to proteolytic degradation. Secondly, the in vitro oxidationof the individually expressed and purified H and L chains to produce theactive di-chain is very inefficient, and leads to low yields of activetoxin and the production of many inactive incorrectly folded or oxidizedforms. The purification of the correctly folded and oxidized H and Lchain-containing toxin is difficult, as is its separation from theseinactive forms and the unreacted separate H and L chains.

It would therefore be useful and advantageous to express clostridialneurotoxins as inactive (or less active) single-chain forms, toeliminate the need for the time-consuming and inefficient reconstitutionof the constituent chains, to maintain solubility of the protein chains,to reduce protein misfolding and consequent susceptibility to proteaseattack, to improve toxin yield, and/or to provide a simple method forthe purification of the toxin.

Additionally, it would be useful to engineer these toxins to providesingle-chain, modified neurotoxin molecules having novel therapeuticproperties and/or longer duration of action, or toxic or non-toxic formsfor use as transport molecules capable of delivering a therapeuticmoiety to nerve or other cell types. By expressing such proteins as asingle-chain, the yield and purification of the engineered proteinswould be vastly improved.

SUMMARY OF THE INVENTION

The present invention is directed to recombinant and isolated proteinscomprising a functional binding domain, translocation domain, andtherapeutic domain in which such proteins also include an amino acidsequence that is susceptible to specific cleavage in vitro followingexpression as a single-chain. Such proteins may include clostridialneurotoxins and derivatives thereof, such as those proteins disclosed inDolly et al., Modified Clostridial Toxins for Use as Transport Proteins,International Patent Publication WO 95/32738 (Dec. 7, 1995); and Fosteret al., Clostridial Toxin Derivatives Able to Modify Peripheral SensoryAfferent Functions, U.S. Pat. No. 5,989,545 (Nov. 23, 1999), bothincorporated by reference herein.

In one embodiment of the invention the protein comprises the functionaldomains of a clostridial neurotoxin H chain and some or all of thefunctions of a clostridial neurotoxin L chain in a single polypeptidechain, and having an inserted proteolytic cleavage site located betweenthe H domain and the L domain by which the single-chain protein may becleaved to produce the individual chains, preferably covalently linkedby a disulfide linkage. The invention also includes methods of makingsuch proteins and expressing them within a cell, as well as nucleic acidvectors for the transfer and expression of the nucleotide sequenceregions encoding such proteins and cells containing such vectors. Theproteolytic cleavage sites comprise amino acid sequences that areselectively recognized and cleaved by a specific enzyme.

In a preferred aspect of the invention, the expressed single-chainproteins comprise the biologically active domains of the H chain and Lchain of a clostridial neurotoxin. Scission at the internal proteolyticcleavage site separating the chain domains thus results in theactivation of a neurotoxin having full activity.

In another aspect of the invention the single-chain proteins comprise abinding domain targeted to a cell receptor other than one borne by amotor neuron. Such a binding domain may specific bind to, for example, asensory afferent neuron, or to a non-neuronal cell type or tissue, suchas pancreatic acinar cells. The single-chain proteins will contain atranslocation domain similar to that of clostridial neurotoxins, and atherapeutic moiety. The therapeutic moiety may be a clostridialneurotoxin light chain, or may be a different therapeutic moiety such asan enzyme, a transcribable nucleotide sequence, growth factor, anantisense nucleotide sequence and the like.

Preferably, the toxins and toxin-based proteins of the present inventionwill be tailored to contain an additional amino acid sequence comprisinga binding tag able to bind a target compound at sufficiently highefficiency to facilitate rapid isolation of the toxin protein. Proteinscontaining such binding sites are many and well known to those of skillin the art, and may comprise, without limitation, monoclonal antibodies,maltose binding protein, glutathione-S-transferase, protein A, a His₆tag, and the like.

Because such proteins exhibit binding selectivity to a certain compoundor compound type, the target compound may be immobilized to a solidsupport, including without limitation, a chromotography resin ormicrotiter well and used for affinity purification of the modifiedtoxin. The toxin molecule can then be eluted by standard methods, suchas through the use of a high salt solution or specific antagonist.

To minimize the safety risk associated with handling neurotoxin, thetoxins of the this aspect of the present invention are expressed astheir low activity (or inactive) single-chain proforms, then, by acarefully controlled proteolytic reaction in vitro, they are activated,preferably to the same potency level as the native neurotoxin from whichthey were derived. To improve the efficiency and rate of proteolyticcleavage the engineered proteolytic cleavage sites can be designed tooccur in a specially-designed loop between the H and L portions of thesingle amino acid chain that promotes accessibility of the protease tothe holotoxin substrate.

To reduce the risk of unintentional activation of the toxin by human orcommonly encountered proteases, the amino acid sequences of the cleavagesite are preferably designed to have a high degree of specificity toproteolytic enzymes which do not normally occur in humans (as eitherhuman proteases or occurring in part of the foreseeable human fauna andflora). A non-exclusive list of examples of such proteases includes aprotease isolated or derived from a non-human Enterokinase, like bovineenterokinase, a protease isolated or derived from plant legumain, aprotease isolated or derived from plant papain, such as, e.g., like fromCarica papaya, a protease isolated or derived from insect papain, likefrom the silkworm Sitophilus zeamatus, a protease isolated or derivedfrom crustacian papain, a protease isolated or derived from Tobacco etchvirus (TEV), a protease isolated or derived from a Tobacco Vein MottlingVirus (TVMV), a protease isolated or derived from Bacillusamyliquifaciens, such as, e.g., subtilisin and GENENASE®, a proteaseisolated or derived from 3c protease from human rhinovirus (HRV), suchas, e.g., PRESCISSION®, a protease isolated or derived from 3c proteasefrom human enteroviruses (HEV), and a protease isolated or derived froma non-human Caspase 3.

In an aspect of the invention the single-chain polypeptide is anisolated polypeptide. By “isolated” is meant removed from its naturalenvironment. For example, for a protein expressed within the cell,isolation includes preparation of a cell lysate as well as subsequentpurification steps. A protein expressed extracellularly may be isolatedby, for example, separation of the supernatant from the cells as well asany subsequent purification steps.

In another aspect of the invention the interchain loop region of the C.botulinum subtype E neurotoxin, which is normally resistant toproteolytic nicking in the bacterium and mammals, is modified to includethe inserted proteolytic cleavage site, and this loop region used as theinterchain loop region in the single-chain toxin or modified toxinmolecules of the present invention. It is believed that using the loopfrom C. botulinum subtype E will stabilize the unnicked toxin moleculein vivo, making it resistant to undesired cleavage until activatedthrough the use of the selected protease.

In yet another aspect of the invention compositions are contemplatedcomprising recombinant forms of BoNT/E expressed as a single-chainpolypeptide.

In still another aspect contemplate recombinant chimeric and/or modifiedtoxin derivatives expressed as a single-chain polypeptide. Suchpolypeptide may be molecular transporters, such as, without limitation,those disclosed in Dolly et al., European Patent Specification EP 0 760681 B1, incorporated by reference herein.

In a further aspect the invention includes neurotoxin derivativescomprising at least a portion of a light chain from one clostridialneurotoxin or subtype thereof, and at least a portion of a heavy chainfrom another neurotoxin or neurotoxin subtype, as well as methods fortheir production. In one embodiment the hybrid neurotoxin may containthe entire light chain of a light chain from one neurotoxin subtype andthe heavy chain from another neurotoxin subtype. In another embodiment,a chimeric neurotoxin derivative may contain a portion (e.g., thebinding domain) of the heavy chain of one neurotoxin subtype, withanother portion of the heavy chain being from another neurotoxinsubtype. Similarly or alternatively, the therapeutic element maycomprise light chain portions from different neurotoxins.

Such hybrid or chimeric neurotoxin derivatives are useful, for example,as a means of delivering the therapeutic benefits of such neurotoxins topatients who are immunologically resistant to a given neurotoxinsubtype, to patients who may have a lower than average concentration ofreceptors to a given neurotoxin heavy chain binding moiety, or topatients who may have a protease-resistant variant of the membrane orvesicle toxin substrate (e.g., SNAP-25, VAMP and syntaxin). Creation ofrecombinant chimeric or hybrid neurotoxin derivatives having a lightchain with different substrate would permit such patients to respond toneurotoxin therapy.

With regard to immunological resistance, it is known that mostneurotoxin epitopes exist on the heavy chain portion of the toxin. Thusif a patient has neutralizing antibodies to, for example BoNT/A, achimeric neurotoxin containing the heavy chain from BoNT/E and the lightchain from BoNT/A (which has a longer duration of therapeutic activitythan other neurotoxin light chains) would overcome this resistance.Likewise if the patient has few cell surface receptors for BoNT/A, thechance are great that the same patient would have adequate receptors toanother BoNT subtype. By creating a hybrid or chimeric neurotoxin (suchas one containing at least a portion of a heavy chain selected from thegroup consisting of HC_(A), HC_(B), HC_(C1), HC_(D), HC_(E), HC_(F), andHC_(G) and a at least a portion of a light chain selected from adifferent clostridial neurotoxin subtype, said light chain beingselected from the group consisting of LC_(A), LC_(B), LC_(C1), LC_(D),LC_(E), LC_(F), and LC_(G)) combining the heavy chain of that subtypewith the most therapeutically appropriate light chain (for example, theBoNT/A light chain) the patient could better respond to neurotoxintherapy.

Another advantage of the hybrid or chimeric neurotoxin derivativesdescribed above is related to the fact that certain of the light chains(e.g., LC_(A)) have a long duration of action, others having a shortduration of action (e.g., LC_(E) and LC_(F)) while still others have anintermediate duration of activity (e.g., LC_(B)). Thus, hybrid andchimeric neurotoxins represent second and third generation neurotoxindrugs in which the neurotoxin activity may be tailored to a specifictherapeutic need or condition, with different drugs having differentactivities, substrate specificities or duration of activity.

Such hybrid or chimeric neurotoxins would also be useful in treating apatient (such as a soldier or laboratory worker) who has been inoculatedwith the pentavalent BoNT vaccine. Such vaccines do not contain BoNT/F;thus, combining the appropriate light chain with the BoNT/F heavy chainwould create a therapeutic agent which is effective in such a patientwhere current therapeutic neurotoxins may not be.

The same strategy may be useful in using derivatives of clostridialneurotoxins with a therapeutic moiety other than an active neurotoxinlight chain. As the heavy chain of such an agent would be derived from aneurotoxin, it may be advantageous to use a lesser known, or rarer heavychain to avoid resistance mechanisms neutralizing the effectiveness ofthe therapeutic neurotoxin derivative.

By the same token, the binding moiety may be one other than a bindingmoiety derived from a clostridial neurotoxin heavy chain, thus providinga targeting function to cell types other than motor neurons.

Also included herein are methods for the construction, expression, andpurification of such molecules in high yield as biologically activeentities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagrammatic view of the single-chain TeNT construct inplasmid pTrcHisA and the nucleotide sequence of the junction region.

FIG. 1B shows the and amino acid sequence connecting the carboxylterminus of the L chain and the amino terminus of the H chain and anengineered loop region containing an enterokinase cleavage site.

FIG. 2A is a representation of a Western blot of an SDS-PAGE gel of cellextracts of E. coli JM 109 transformants containing 2 differentrecombinant single-chain toxins, either before or after induction ofplasmid protein expression with IPTG. The antibody used for detection isan anti-His₆ monoclonal antibody.

FIG. 2B is a Western blot of IPTG-induced cell extracts from cellstransformed with the E234A construct.

FIG. 3A shows the results of an experiment in which affinity purifiedrecombinant single-chain (SC) TeNT is nicked with enterokinase, thenseparated using SDS-PAGE and visualized using Commassie Brilliant Blueunder reducing and non-reducing conditions.

FIG. 3B shows the results of an experiment in which affinity purifiedrecombinant single-chain (SC) TeNT is nicked with enterokinase, thenseparated using SDS-PAGE under reducing and non-reducing conditions andsubjected to a Western blot using anti TeNT heavy chain antibody.

FIG. 4 is a plot of the degree of paralysis induced in a nerve/musclepreparation in vitro using native TeNT, and recombinant single-chainneurotoxin before, and after nicking as a function of time.

FIG. 5 is a depiction of the peptide fragments generated upon incubationof the recombinant single-chain TeNT with trypsin and Arg C protease,and deduction, from the N-terminal sequences of one of the resultingfragments, of the amino acid sequence recognized by these agents.

FIG. 6 shows the digestion of unnicked SC WT TeNT and SC R496G TeNT withvarious concentrations of trypsin.

FIG. 7 shows the inhibitory effect upon TeNT stimulated inhibition ofCa⁺⁺-dependent neurotransmitter release of preincubating cerebellarcells with the E234A mutant TeNT.

FIG. 8 shows the effect upon Ca⁺⁺-dependent neurotransmitter release ofcerebellar neurons upon exposure to native, recombinant E234A mutantsingle-chain, and the recombinant R496G mutant single-chain TeNT.

FIG. 9 shows the inhibitory effect upon TeNT-stimulated paralyticactivity of preincubating mouse hemi diaphrams with the E234A mutantTeNT.

FIG. 10 shows the scheme for construction of a plasmid encodingsingle-chain BoNT/E, and an agarose gel electrophoretogram of the PCRfragment obtained during the construction of the plasmid.

FIG. 11 shows the scheme for construction of a plasmid encoding theE212Q proteolytically inactive single-chain BoNT/E mutant, and anagarose gel electrophoretogram of the inverse PCR fragment obtainedduring the construction of the plasmid.

FIG. 12 shows the expression and purification scheme for recombinantsingle-chain BoNT/E, and a SDS-PAGE electrophoretogram and Western blotof the purification fractions.

FIG. 13 shows SDS-PAGE electrophoretograms under reducing andnon-reducing conditions of native recombinant unnicked, and recombinantnicked BoNT/E, and Western Blots directed towards the heavy and lightchains of the toxin.

FIG. 14 shows the results of incubating native BoNT/E, recombinantnicked and un-nicked BoNT/E, and the E212Q mutant with a GST-SNAP-25[140-205] protease substrate.

FIG. 15 shows the effect upon Ca++-dependent glutamate release ofincubating cerebellar cells with native BoNT/E, un-nicked recombinantsingle-chain BoNT/E, and nicked recombinant single-chain BoNT/E.

FIG. 16A shows the effects on muscle tension of incubating mousephrenic-nerve hemi-diaphragms with 0.2 nM recombinant nicked BoNT/E (∘)or 0.2 nM native BoNT/E (□).

FIG. 16B shows the effects on muscle tension of incubating mousephrenic-nerve hemi-diaphragms with 1 nM recombinant un-nicked (∘), 1 nMrecombinant nicked (●) or 0.05 nM recombinant nicked (∇) BoNT/E.

FIG. 17 shows the attenuation of paralytic activity on mousephrenic-nerve hemi-diaphragms of preincubation with the inactive E212Qmutant prior to exposure to native nicked BoNT/E toxin.

FIG. 18 shows a schematic of the current paradigm of neurotransmitterrelease and Clostridial toxin intoxication in a central and peripheralneuron. FIG. 18A shows a schematic for the neurotransmitter releasemechanism of a central and peripheral neuron. The release process can bedescribed as comprising two steps: 1) vesicle docking, where thevesicle-bound SNARE protein of a vesicle containing neurotransmittermolecules associates with the membrane-bound SNARE proteins located atthe plasma membrane; and 2) neurotransmitter release, where the vesiclefuses with the plasma membrane and the neurotransmitter molecules areexocytosed. FIG. 18B shows a schematic of the intoxication mechanism fortetanus and botulinum toxin activity in a central and peripheral neuron.This intoxication process can be described as comprising four steps: 1)receptor binding, where a Clostridial toxin binds to a Clostridialreceptor system and initiates the intoxication process; 2) complexinternalization, where after toxin binding, a vesicle containing thetoxin/receptor system complex is endocytosed into the cell; 3) lightchain translocation, where multiple events are thought to occur,including, e.g., changes in the internal pH of the vesicle, formation ofa channel pore comprising the HN domain of the Clostridial toxin heavychain, separation of the Clostridial toxin light chain from the heavychain, and release of the active light chain and 4) enzymatic targetmodification, where the activate light chain of Clostridial toxinproteolytically cleaves its target SNARE substrate, such as, e.g.,SNAP-25, VAMP or Syntaxin, thereby preventing vesicle docking andneurotransmitter release.

FIG. 19 shows the domain organization of naturally-occurring Clostridialtoxins. The single-chain form depicts the amino to carboxyl linearorganization comprising an enzymatic domain, a translocation domain, anda binding domain. The di-chain loop region located between thetranslocation and enzymatic domains is depicted by the double SSbracket. This region comprises an endogenous di-chain loop proteasecleavage site that upon proteolytic cleavage with a naturally-occurringprotease, such as, e.g., an endogenous Clostridial toxin protease or anaturally-occurring protease produced in the environment, converts thesingle-chain form of the toxin into the di-chain form. Above thesingle-chain form, the H_(CC) region of the Clostridial toxin bindingdomain is depicted. This region comprises the β-trefoil domain whichcomprises in a amino to carboxyl linear organization an α-fold, a β4/β5hairpin turn, β-fold, a β8/β9 hairpin turn and a γ-fold.

FIG. 20 shows modified Clostridial toxins with an enhanced targetingdomain located at the amino terminus of the modified toxin. FIG. 20Adepicts the single-chain polypeptide form of a modified Clostridialtoxin with an amino to carboxyl linear organization comprising a bindingelement, a translocation element, a di-chain loop region comprising anexogenous protease cleavage site (P), and a therapeutic element. Uponproteolytic cleavage with a P protease, the single-chain form of thetoxin is converted to the di-chain form. FIG. 20B depicts the singlepolypeptide form of a modified Clostridial toxin with an amino tocarboxyl linear organization comprising a binding element, a therapeuticelement, a di-chain loop region comprising an exogenous proteasecleavage site (P), and a translocation element. Upon proteolyticcleavage with a P protease, the single-chain form of the toxin isconverted to the di-chain form.

FIG. 21 shows modified Clostridial toxins with an enhanced targetingdomain located between the other two domains. FIG. 21A depicts thesingle polypeptide form of a modified Clostridial toxin with an amino tocarboxyl linear organization comprising a therapeutic element, adi-chain loop region comprising an exogenous protease cleavage site (P),a binding element, and a translocation element. Upon proteolyticcleavage with a P protease, the single-chain form of the toxin isconverted to the di-chain form. FIG. 21B depicts the single polypeptideform of a modified Clostridial toxin with an amino to carboxyl linearorganization comprising a translocation element, a di-chain loop regioncomprising an exogenous protease cleavage site (P), a binding element,and a therapeutic element. Upon proteolytic cleavage with a P protease,the single-chain form of the toxin is converted to the di-chain form.FIG. 21C depicts the single polypeptide form of a modified Clostridialtoxin with an amino to carboxyl linear organization comprising atherapeutic element, a binding element, a di-chain loop regioncomprising an exogenous protease cleavage site (P), and a translocationelement. Upon proteolytic cleavage with a P protease, the single-chainform of the toxin is converted to the di-chain form. FIG. 21D depictsthe single polypeptide form of a modified Clostridial toxin with anamino to carboxyl linear organization comprising a translocationelement, a binding element, a di-chain loop region comprising anexogenous protease cleavage site (P), and a therapeutic element. Uponproteolytic cleavage with a P protease, the single-chain form of thetoxin is converted to the di-chain form.

FIG. 22 shows modified Clostridial toxins with an enhanced targetingdomain located at the carboxyl terminus of the modified toxin. FIG. 22Adepicts the single polypeptide form of a modified Clostridial toxin withan amino to carboxyl linear organization comprising a therapeuticelement, a di-chain loop region comprising an exogenous proteasecleavage site (P), a translocation element, and a binding element. Uponproteolytic cleavage with a P protease, the single-chain form of thetoxin is converted to the di-chain form. FIG. 22B depicts the singlepolypeptide form of a modified Clostridial toxin with an amino tocarboxyl linear organization comprising a translocation element, adi-chain loop region comprising an exogenous protease cleavage site (P),a therapeutic element, and a binding element. Upon proteolytic cleavagewith a P protease, the single-chain form of the toxin is converted tothe di-chain form.

DETAILED DESCRIPTION OF THE INVENTION

Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct types ofBotulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, /B, /E and /F), animals (BoNT/C1 and/D), or isolated from soil (BoNT/G). BoNTs possess approximately 35%amino acid identity with each other and share the same functional domainorganization and overall structural architecture. It is recognized bythose of skill in the art that within each type of Clostridial toxinthere can be subtypes that differ somewhat in their amino acid sequence,and also in the nucleic acids encoding these proteins. For example,there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4, with specific subtypes showing approximately 89% amino acididentity when compared to another BoNT/A subtype. While all seven BoNTserotypes have similar structure and pharmacological properties, eachalso displays heterogeneous bacteriological characteristics. Incontrast, tetanus toxin (TeNT) is produced by a uniform group of C.tetani. Two other species of Clostridia, C. baratii and C. butyricum,also produce toxins, BaNT and BuNT respectively, which are similar toBoNT/F and BoNT/E, respectively.

Each mature di-chain molecule comprises three functionally distinctdomains: 1) an enzymatic domain located in the LC that includes ametalloprotease region containing a zinc-dependent endopeptidaseactivity which specifically targets core components of theneurotransmitter release apparatus; 2) a translocation domain containedwithin the amino-terminal half of the HC (H_(N)) that facilitatesrelease of the LC from intracellular vesicles into the cytoplasm of thetarget cell; and 3) a binding domain found within the carboxyl-terminalhalf of the HC (H_(C)) that determines the binding activity and bindingspecificity of the toxin to the receptor complex located at the surfaceof the target cell. The H_(C) domain comprises two distinct structuralfeatures of roughly equal size that indicate function and are designatedthe H_(CN) and H_(CC) subdomains. Table 1 gives approximate boundaryregions for each domain found in exemplary Clostridial toxins. TABLE 1Clostridial Toxin Reference Sequences and Regions Toxin SEQ ID NO: LCH_(N) H_(C) BoNT/A 1 M1-K448 A449-K871 N872-L1296 BoNT/B 2 M1-K441A442-S858 E859-E1291 BoNT/C1 3 M1-K449 T450-N866 N867-E1291 BoNT/D 4M1-R445 D446-N862 S863-E1276 BoNT/E 5 M1-R422 K423-K845 R846-K1252BoNT/F 6 M1-K439 A440-K864 K865-E1274 BoNT/G 7 M1-K446 S447-S863N864-E1297 TeNT 8 M1-A457 S458-V879 I880-D1315 BaNT 9 M1-K431 N432-I857I858-E1268 BuNT 10 M1-R422 K423-I847 Y1086-K1251

The binding, translocation and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby Clostridial toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of serotype orsubtype. Although the applicants have no wish to be limited by thefollowing description, the intoxication mechanism can be described ascomprising at least four steps: 1) receptor binding, 2) complexinternalization, 3) light chain translocation, and 4) enzymatic targetmodification (see FIG. 18). The process is initiated when the H_(C)domain of a Clostridial toxin binds to a toxin-specific receptor systemlocated on the plasma membrane surface of a target cell. The bindingspecificity of a receptor complex is thought to be achieved, in part, byspecific combinations of gang liosides and protein receptors that appearto distinctly comprise each Clostridial toxin receptor complex. Oncebound, the toxin/receptor complexes are internalized by endocytosis andthe internalized vesicles are sorted to specific intracellular routes.The translocation step appears to be triggered by the acidification ofthe vesicle compartment. This process seems to initiate two importantpH-dependent structural rearrangements that increase hydrophobicity andpromote formation di-chain form of the toxin. Once activated, lightchain endopeptidase of the toxin is released from the intracellularvesicle into the cytosol where it appears to specifically targets one ofthree known core components of the neurotransmitter release apparatus.These core proteins, vesicle-associated membrane protein(VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa(SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking andfusion at the nerve terminal and constitute members of the solubleN-ethylmaleimide-sensitive factor-attachment protein-receptor (SNARE)family. BoNT/A and BoNT/E cleave SNAP-25 in the carboxyl-terminalregion, releasing a nine or twenty-six amino acid segment, respectively,and BoNT/C1 also cleaves SNAP-25 near the carboxyl-terminus. Thebotulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and tetanustoxin, act on the conserved central portion of VAMP, and release theamino-terminal portion of VAMP into the cytosol. BoNT/C1 cleavessyntaxin at a single site near the cytosolic membrane surface. Theselective proteolysis of synaptic SNAREs accounts for the block ofneurotransmitter release caused by Clostridial toxins in vivo. The SNAREprotein targets of Clostridial toxins are common to exocytosis in avariety of non-neuronal types; in these cells, as in neurons, lightchain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau etal., How Botulinum and Tetanus Neurotoxins Block NeurotransmitterRelease, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,Botulinum and Tetanus Neurotoxins: Structure, Function and TherapeuticUtility, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna Lalli etal., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11 (9)Trends Microbiol. 431-437, (2003).

Clostridial toxins are each translated as a single-chain polypeptide ofapproximately 150 kDa that is subsequently cleaved by proteolyticscission within a disulfide loop by a naturally-occurring protease (FIG.18). This cleavage occurs within the discrete di-chain loop regioncreated between two cysteine residues that form a disulfide bridge. Thisposttranslational processing yields a di-chain molecule comprising anapproximately 50 kDa light chain (LC) and an approximately 100 kDa heavychain (HC) held together by the single disulfide bond and non-covalentinteractions between the two chains. The naturally-occurring proteaseused to convert the single-chain molecule into the di-chain is currentlynot known. In some bacterial serotypes, such as, e.g., a BoNT/A, aBoNT/B proteolytic, a BoNT/F proteolytic, a BaNT proteolytic strain, ora TeNT, the naturally-occurring protease is produced endogenously by thebacteria serotype and cleavage occurs within the cell before the toxinis release into the environment. However, in other bacterial serotypes,such as, e.g., a BoNT/B nonproteolytic, a BoNT/C1, a BoNT/D, a BoNT/E, aBoNT/F nonproteolytic, a BoNT/G, a BaNT nonproteolytic, or a BuNT, thebacterial strain appears not to produce appreciable amounts of anendogenous protease capable of converting the single-chain form of thetoxin into the di-chain form. In these situations, the toxin is releasedfrom the cell as a single-chain toxin which is subsequently convertedinto the di-chain form by a naturally-occurring protease found in theenvironment.

The compositions and methods of the present invention involve modifiedneurotoxins, their synthesis and use. Di-chain neurotoxins that arenormally activated by scission of a single-chain polypeptide byindigenous proteases can be modified at the nucleic acid level byalteration or removal of the nucleotide sequence encoding the indigenousprotease cleavage site and insertion of a nucleotide sequence encodinganother different proteolytic cleavage site resistant to cleavage byhost cell or human proteases. The inserted amino acid sequence isdesigned to be cleaved in vitro through the use of a cleaving agentchosen in advance of expression that is, absent from both human and hostcell tissue.

The amino acid sequences recognized by many proteases, and theircleavage specificity are well-known to those of skill in the art. Thus,both the design of a specific proteolytic cleavage site in the loopregion between the L and H chain portions of the single-chain toxin andthe modification of incidental protease sites in the polypeptide to beprotease-resistant is a routine matter of comparing the specificity andrecognition sequences for various proteins. In the first case, thespecificity of a candidate proteolytic site need not be totallyexclusive, but merely needs to exclude cleavage sites for human and/orhost cell proteases that might be present during the handling, storageand purification of the single-chain neurotoxin. Of course, it ispreferable that the protease site is as specific as possible. In thelatter case, the modification of the proteolytic cleavage site need onlybe sufficient to render the site resistant to the activator protease andto human and host cell proteases.

As mentioned above, a Clostridial toxin is converted from a singlepolypeptide form into a di-chain molecule by proteolytic cleavage. Whilethe naturally-occurring protease is currently not known, cleavage occurswithin the di-chain loop region between the two cysteine residues thatform the disulfide bridge (Table 2). As used herein, the term “di-chainloop region” means the amino acid sequence of a Clostridial toxincontaining a protease cleavage site used to convert the single-chainpolypeptide form of a Clostridial toxin into the di-chain form.Non-limiting examples of a Clostridial toxin di-chain loop region,include, a di-chain loop region of BoNT/A comprising SEQ ID NO: 11; adi-chain loop region of BoNT/B comprising SEQ ID NO: 12; a di-chain loopregion of BoNT/C1 comprising SEQ ID NO: 13; a di-chain loop region ofBoNT/D comprising SEQ ID NO: 14; a di-chain loop region of BoNT/Ecomprising SEQ ID NO: 15; a di-chain loop region of BoNT/F comprisingSEQ ID NO: 16; a di-chain loop region of BoNT/G comprising SEQ ID NO:17; a di-chain loop region of TeNT comprising SEQ ID NO: 18, a di-chainloop region of BaNT comprising SEQ ID NO: 19, and a di-chain loop regionof BuNT comprising SEQ ID NO: 20 (Table 2). TABLE 2 Di-chain Loop Regionof Clostridial Toxins Di-Chain Loop Region Including a Di-Chain ToxinSEQ ID NO: Protease Cleavage Site BoNT/A 11CVRGIITSKTKSLDKGYNK*----ALNDLC BoNT/B 12 CKSVK*-------------------APGICBoNT/C1 13 CHKAIDGRSLYNK*------------TLDC BoNT/D 14CLRLTKNSR*---------------DDSTC BoNT/E 15 CKNIVSVKGIR*--------------KSICBoNT/F 16 CKSVIPRKGTK*------------APPRLC BoNT/G 17CKPVMYKNTGK*--------------SEQC TeNT 18 CKKIIPPTNIRENLYNRTA*SLTDLGGELCBaNT 19 CKSIVSKKGTK*--------------NSLC BuNT 20CKNIVSVKGIR*--------------KSICThe amino acid sequence displayed are as follows: BoNT/A, residues430-454 of SEQ ID NO: 1; BoNT/B, residues 437-446 of SEQ ID NO: 2;BoNT/C1, residues 437-453 of SEQ ID NO: 3; BoNT/D, residues 437-450 ofSEQ ID NO: 4; BoNT/E, residues 412-426 of SEQ ID NO: 5; BoNT/F, residues429-445 of SEQ ID NO: 6; BoNT/G, residues 436-450 of SEQ ID NO: 7; TeNT,residues 439-467 of SEQ ID NO: 8; BaNT, residues 421-435 of SEQ ID NO:9; and BuNT, residues 412-426 of SEQ ID NO: 10.An asterisks (*) indicates the peptide bond of the P₁—P₁′ cleavage sitethat is believed to be cleaved by a Clostridial toxin di-chain loopprotease.

The inserted amino acid sequence may be chosen to confer susceptibilityto a chemical agent capable of cleaving peptide bonds, such as cyanogenbromide. However, and much more preferably, the encoded amino acidsequence may comprise a proteolytic cleavage site highly specific for aselected protease. The selected protease may be any protease thatrecognizes a specific amino acid sequence and cleaves a peptide bondnear or at that location, but the selected protease is very preferablynot a human protease such as, e.g., human trypsin, chymotrypsin orpepsin, or a protease expressed in the host cell. Moreover, the selectedprotease does not recognize the same amino acid sequence as theendogenous protease (i.e., the naturally-occurring di-chain loopprotease cleavage site). Finally, the selected protease should not beone expressed by the host cell that contains the plasmid encoding therecombinant neurotoxin. Any non-human protease recognizing a relativelyrare amino acid sequence may be used, provided that the amino acidrecognition sequence is also known. Examples of proteases to be selectedas activators may include any of the following, without limitation: aprotease isolated or derived from non-human Enterokinase, such as, e.g.,a bovine enterokinase, a protease isolated or derived from plantlegumain, a protease isolated or derived from plant papain, such as,e.g., like from Carica papaya, a protease isolated or derived frominsect papain, like from the silkworm Sitophilus zeamatus, a proteaseisolated or derived from crustacian papain, a protease isolated orderived from Tobacco etch virus (TEV), a protease isolated or derivedfrom a Tobacco Vein Mottling Virus (TVMV), a protease isolated orderived from Bacillus amyliquifaciens, such as, e.g., subtilisin andGENENASE®, a protease isolated or derived from 3c protease from humanrhinovirus (HRV), such as, e.g., PRESCISSION®, a protease isolated orderived from 3c protease from human enteroviruses (HEV) and a proteaseisolated or derived from a non-human Caspase 3, such as, e.g., a mouseCaspase 3.

In another aspect of the invention, a modified Clostridial toxincomprises, in part, an exogenous protease cleavage site within adi-chain loop region. As used herein, the term “exogenous proteasecleavage site” is synonymous with a “non-naturally occurring proteasecleavage site” or “non-native protease cleavage site” and means aprotease cleavage site that is not normally present in a di-chain loopregion from a naturally occurring Clostridial toxin, with the provisothat the exogenous protease cleavage site is not a human proteasecleavage site or a protease cleavage site that is susceptible to aprotease being expressed in the host cell that is expressing a constructencoding an activatable polypeptide disclosed in the presentspecification. It is envisioned that any and all exogenous proteasecleavage sites can be used to convert the single-chain polypeptide formof a Clostridial toxin into the di-chain form are useful to practiceaspects of the present invention. Non-limiting examples of exogenousprotease cleavage sites include, e.g., a plant papain cleavage site, aninsect papain cleavage site, a crustacian papain cleavage site, anon-human enterokinase cleavage site, a human rhinovirus 3C proteasecleavage site, human enterovirus 3C protease cleavage site, a tobaccoetch virus (TEV) protease cleavage site, a Tobacco Vein Mottling Virus(TVMV) cleavage site, a subtilisin cleavage site, a hydroxylaminecleavage site, or a non-human Caspase 3 cleavage site.

It is envisioned that an exogenous protease cleavage site of any and alllengths can be useful in aspects of the present invention with theproviso that the exogenous protease cleavage site is capable of beingcleaved by its respective protease. Thus, in aspects of this embodiment,an exogenous protease cleavage site can be, e.g., at least 6 amino acidsin length, at least 7 amino acids in length, at least 8 amino acids inlength, at least 9 amino acids in length, at least 10 amino acids inlength, at least 15 amino acids in length, at least 20 amino acids inlength, at least 25 amino acids in length, at least 30 amino acids inlength, at least 40 amino acids in length, at least 50 amino acids inlength or at least 60 amino acids in length. In other aspects of thisembodiment, an exogenous protease cleavage site can be, e.g., at most 6amino acids in length, at most 7 amino acids in length, at most 8 aminoacids in length, at most 9 amino acids in length, at most 10 amino acidsin length, at most 15 amino acids in length, at most 20 amino acids inlength, at most 25 amino acids in length, at most 30 amino acids inlength, at most 40 amino acids in length, at most 50 amino acids inlength or at most 60 amino acids in length.

In an embodiment, an exogenous protease cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In aspects of thisembodiment, a modified Clostridial toxin comprises an exogenous proteasecleavage site comprises, e.g., a plant papain cleavage site, an insectpapain cleavage site, a crustacian papain cleavage site, a non-humanenterokinase protease cleavage site, a Tobacco Etch Virus proteasecleavage site, a Tobacco Vein Mottling Virus protease cleavage site, ahuman rhinovirus 3C protease cleavage site, a human enterovirus 3Cprotease cleavage site, a subtilisin cleavage site, a hydroxylaminecleavage site, a SUMO/ULP-1 protease cleavage site, and a non-humanCaspase 3 cleavage site. In other aspects of this embodiment, anexogenous protease cleavage site is located within the di-chain loop of,e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, amodified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modifiedBoNT/G, a modified TeNT, a modified BaNT, or a modified BuNT.

In an aspect of this embodiment, an exogenous protease cleavage site cancomprise, e.g., a non-human enterokinase cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In other aspects ofthe embodiment, an exogenous protease cleavage site can comprise, e.g.,a bovine enterokinase protease cleavage site located within the di-chainloop of a modified Clostridial toxin. In other aspects of theembodiment, an exogenous protease cleavage site can comprise, e.g., abovine enterokinase protease cleavage site located within the di-chainloop of a modified Clostridial toxin comprises SEQ ID NO: 21. In stillother aspects of this embodiment, a bovine enterokinase proteasecleavage site is located within the di-chain loop of, e.g., a modifiedBoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, amodified BoNT/E, a modified BoNT/F, a modified BoNT/G, a modified TeNT,a modified BaNT, or a modified BuNT.

In another aspect of this embodiment, an exogenous protease cleavagesite can comprise, e.g., a Tobacco Etch Virus protease cleavage site islocated within the di-chain loop of a modified Clostridial toxin. Inother aspects of the embodiment, an exogenous protease cleavage site cancomprise, e.g., a Tobacco Etch Virus protease cleavage site locatedwithin the di-chain loop of a modified Clostridial toxin comprises theconsensus sequence E-P5-P4-Y-P2-Q*-G (SEQ ID NO: 22) orE-P5-P4-Y-P2-Q*-S (SEQ ID NO: 23), where P2, P4 and P5 can be any aminoacid. In other aspects of the embodiment, an exogenous protease cleavagesite can comprise, e.g., a Tobacco Etch Virus protease cleavage sitelocated within the di-chain loop of a modified Clostridial toxincomprises SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32 or SEQ ID NO: 33. In still other aspects of this embodiment, aTobacco Etch Virus protease cleavage site is located within the di-chainloop of, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1,a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modifiedBoNT/G, a modified TeNT, a modified BaNT, or a modified BuNT.

In another aspect of this embodiment, an exogenous protease cleavagesite can comprise, e.g., a Tobacco Vein Mottling Virus protease cleavagesite is located within the di-chain loop of a modified Clostridialtoxin. In other aspects of the embodiment, an exogenous proteasecleavage site can comprise, e.g., a Tobacco Vein Mottling Virus proteasecleavage site located within the di-chain loop of a modified Clostridialtoxin comprises the consensus sequence P6-P5-V-R-F-Q*-G (SEQ ID NO: 34)or P6-P5-V-R-F-Q*-S (SEQ ID NO: 35), where P5 and P6 can be any aminoacid. In other aspects of the embodiment, an exogenous protease cleavagesite can comprise, e.g., a Tobacco Vein Mottling Virus protease cleavagesite located within the di-chain loop of a modified Clostridial toxincomprises SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39.In still other aspects of this embodiment, a Tobacco Vein Mottling Virusprotease cleavage site is located within the di-chain loop of, e.g., amodified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modifiedBoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G, amodified TeNT, a modified BaNT, or a modified BuNT.

In still another aspect of this embodiment, an exogenous proteasecleavage site can comprise, e.g., a human rhinovirus 3C proteasecleavage site is located within the di-chain loop of a modifiedClostridial toxin. In other aspects of the embodiment, an exogenousprotease cleavage site can comprise, e.g., a human rhinovirus 3Cprotease cleavage site located within the di-chain loop of a modifiedClostridial toxin comprises the consensus sequence P5-P4-L-F-Q*-G-P (SEQID NO: 40), where P4 is G, A, V, L, I, M, S or T and P5 can any aminoacid, with D or E preferred. In other aspects of the embodiment, anexogenous protease cleavage site can comprise, e.g., a human rhinovirus3C protease cleavage site located within the di-chain loop of a modifiedClostridial toxin comprises SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46. In other aspects of theembodiment, an exogenous protease cleavage site can comprise, e.g., ahuman rhinovirus 3C protease located within the di-chain loop of amodified Clostridial toxin that can be cleaved by PRESCISSION®. In stillother aspects of this embodiment, a human rhinovirus 3C proteasecleavage site is located within the di-chain loop of, e.g., a modifiedBoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, amodified BoNT/E, a modified BoNT/F, a modified BoNT/G, a modified TeNT,a modified BaNT, or a modified BuNT.

In yet another aspect of this embodiment, an exogenous protease cleavagesite can comprise, e.g., a subtilisin cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In other aspects ofthe embodiment, an exogenous protease cleavage site can comprise, e.g.,a subtilisin cleavage site located within the di-chain loop of amodified Clostridial toxin comprises the consensus sequenceP6-P5-P4-P3-H*-Y (SEQ ID NO: 47) or P6-P5-P4-P3-Y-H* (SEQ ID NO: 48),where P3, P4 and P5 and P6 can be any amino acid. In other aspects ofthe embodiment, an exogenous protease cleavage site can comprise, e.g.,a subtilisin cleavage site located within the di-chain loop of amodified Clostridial toxin comprises SEQ ID NO: 49, SEQ ID NO: 50, orSEQ ID NO: 51. In other aspects of the embodiment, an exogenous proteasecleavage site can comprise, e.g., a subtilisin cleavage site locatedwithin the di-chain loop of a modified Clostridial toxin that can becleaved by GENENASE®. In still other aspects of this embodiment, asubtilisin cleavage site is located within the di-chain loop of, e.g., amodified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modifiedBoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G, amodified TeNT, a modified BaNT, or a modified BuNT.

In yet another aspect of this embodiment, an exogenous protease cleavagesite can comprise, e.g., a hydroxylamine cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In other aspects ofthe embodiment, an exogenous protease cleavage site can comprise, e.g.,a hydroxylamine cleavage site comprising multiples of the dipeptide N*G.In other aspects of the embodiment, an exogenous protease cleavage sitecan comprise, e.g., a hydroxylamine cleavage site located within thedi-chain loop of a modified Clostridial toxin comprises SEQ ID NO: 52,SEQ ID NO: 53, or SEQ ID NO: 54. In still other aspects of thisembodiment, a hydroxylamine cleavage site is located within the di-chainloop of, e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1,a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modifiedBoNT/G, a modified TeNT, a modified BaNT, or a modified BuNT.

In yet another aspect of this embodiment, an exogenous protease cleavagesite can comprise, e.g., a SUMO/ULP-1 protease cleavage site is locatedwithin the di-chain loop of a modified Clostridial toxin. In otheraspects of the embodiment, an exogenous protease cleavage site cancomprise, e.g., a SUMO/ULP-1 protease cleavage site located within thedi-chain loop of a modified Clostridial toxin comprising the consensussequence G-G*-P1′-P2′-P3′ (SEQ ID NO: 55), where P1′, P2′, and P3′ canbe any amino acid. In other aspects of the embodiment, an exogenousprotease cleavage site can comprise, e.g., a SUMO/ULP-1 proteasecleavage site located within the di-chain loop of a modified Clostridialtoxin comprises SEQ ID NO: 56. In still other aspects of thisembodiment, a SUMO/ULP-1 protease cleavage site is located within thedi-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modifiedBoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, amodified BoNT/G, a modified TeNT, a modified BaNT, or a modified BuNT.

In an aspect of this embodiment, an exogenous protease cleavage site cancomprise, e.g., a non-human Caspase 3 cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In other aspects ofthe embodiment, an exogenous protease cleavage site can comprise, e.g.,a mouse Caspase 3 protease cleavage site located within the di-chainloop of a modified Clostridial toxin. In other aspects of theembodiment, an exogenous protease cleavage site can comprise, e.g., anon-human Caspase 3 protease cleavage site located within the di-chainloop of a modified Clostridial toxin comprises the consensus sequenceD-P3-P2-D*P1′ (SEQ ID NO: 57), where P3 can be any amino acid, with Epreferred, P2 can be any amino acid and P1′ can any amino acid, with Gor S preferred. In other aspects of the embodiment, an exogenousprotease cleavage site can comprise, e.g., a non-human Caspase 3protease cleavage site located within the di-chain loop of a modifiedClostridial toxin comprising SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63. In still otheraspects of this embodiment, a bovine enterokinase protease cleavage siteis located within the di-chain loop of, e.g., a modified BoNT/A, amodified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modifiedBoNT/E, a modified BoNT/F, a modified BoNT/G, a modified TeNT, amodified BaNT, or a modified BuNT.

A di-chain loop region is modified to replace a naturally-occurringdi-chain loop protease cleavage site for an exogenous protease cleavagesite. In this modification, the naturally-occurring di-chain loopprotease cleavage site is made inoperable and thus can not be cleaved byits protease. Only the exogenous protease cleavage site can be cleavedby its corresponding exogenous protease. In this type of modification,the exogenous protease site is operably-linked in-frame to a modifiedClostridial toxin as a fusion protein and the site can be cleaved by itsrespective exogenous protease. Replacement of an endogenous di-chainloop protease cleavage site with an exogenous protease cleavage site canbe a substitution of the sites where the exogenous site is engineered atthe position approximating the cleavage site location of the endogenoussite. Replacement of an endogenous di-chain loop protease cleavage sitewith an exogenous protease cleavage site can be an addition of anexogenous site where the exogenous site is engineered at the positiondifferent from the cleavage site location of the endogenous site, theendogenous site being engineered to be inoperable. The location and kindof protease cleavage site may be critical because certain bindingdomains require a free amino-terminal or carboxyl-terminal amino acid.For example, when a binding domain is placed between two other domains,e.g., see FIG. 22, a criterion for selection of a protease cleavage sitecould be whether the protease that cleaves its site leaves a flush cut,exposing the free amino-terminal or carboxyl-terminal of the bindingdomain necessary for selective binding of the binding domain to itsreceptor.

A naturally-occurring protease cleavage site can be made inoperable byaltering at least the two amino acids flanking the peptide bond cleavedby the naturally-occurring di-chain loop protease. More extensivealterations can be made, with the proviso that the two cysteine residuesof the di-chain loop region remain intact and the region can still formthe disulfide bridge. Non-limiting examples of an amino acid alterationinclude deletion of an amino acid or replacement of the original aminoacid with a different amino acid. Thus, in one embodiment, anaturally-occurring protease cleavage site is made inoperable byaltering the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease. In other aspects of this embodiment, anaturally-occurring protease cleavage site is made inoperable byaltering, e.g., at least three amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease; atleast four amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at least fiveamino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at least six amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least seven amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least eight amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least nine amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least ten amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; at least 15 amino acids including the two amino acids flankingthe peptide bond cleaved by a naturally-occurring protease; or at least20 amino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease.

In still other aspects of this embodiment, a naturally-occurringdi-chain protease cleavage site is made inoperable by altering, e.g., atmost three amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at most fouramino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at most five amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at most six amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; at most seven amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease; atmost eight amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at most nineamino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at most ten amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at most 15 amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; or at most 20 amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease.

It is understood that a modified Clostridial toxin disclosed in thepresent specification can optionally further comprise a flexible regioncomprising a flexible spacer. Non-limiting examples of a flexible spacerinclude, e.g., a G-spacer GGGGS (SEQ ID NO: 64) or an A-spacer EAAAK(SEQ ID NO: 65). A flexible region comprising flexible spacers can beused to adjust the length of a polypeptide region in order to optimize acharacteristic, attribute or property of a polypeptide. Such a flexibleregion is operably-linked in-frame to the modified Clostridial toxin asa fusion protein. As a non-limiting example, a polypeptide regioncomprising one or more flexible spacers in tandem can be use to betterexpose a protease cleavage site thereby facilitating cleavage of thatsite by a protease. As another non-limiting example, a polypeptideregion comprising one or more flexible spacers in tandem can be use tobetter present a binding domain, thereby facilitating the binding ofthat binding domain to its receptor.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise a flexible region comprisinga flexible spacer. In another embodiment, a modified Clostridial toxindisclosed in the present specification can further comprise flexibleregion comprising a plurality of flexible spacers in tandem. In aspectsof this embodiment, a flexible region can comprise in tandem, e.g., atleast 1 G-spacer, at least 2 G-spacers, at least 3 G-spacers, at least 4G-spacers or at least 5 G-spacers. In other aspects of this embodiment,a flexible region can comprise in tandem, e.g., at most 1 G-spacer, atmost 2 G-spacers, at most 3 G-spacers, at most 4 G-spacers or at most 5G-spacers. In still other aspects of this embodiment, a flexible regioncan comprise in tandem, e.g., at least 1 A-spacer, at least 2 A-spacers,at least 3 A-spacers, at least 4 A-spacers or at least 5 A-spacers. Instill other aspects of this embodiment, a flexible region can comprisein tandem, e.g., at most 1 A-spacer, at most 2 A-spacers, at most 3A-spacers, at most 4 A-spacers or at most 5 A-spacers. In another aspectof this embodiment, a modified Clostridial toxin can comprise a flexibleregion comprising one or more copies of the same flexible spacers, oneor more copies of different flexible-spacer regions, or any combinationthereof.

In other aspects of this embodiment, a modified Clostridial toxincomprising a flexible spacer can be, e.g., a modified BoNT/A, a modifiedBoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, amodified BoNT/F, a modified BoNT/G, a modified TeNT, a modified BaNT, ora modified BuNT.

It is envisioned that a modified Clostridial toxin disclosed in thepresent specification can comprise a flexible spacer in any and alllocations with the proviso that modified Clostridial toxin is capable ofperforming the intoxication process. In aspects of this embodiment, aflexible spacer is positioned between, e.g., a therapeutic element and atranslocation element, a therapeutic element and a binding element, atherapeutic element and an exogenous protease cleavage site. In otheraspects of this embodiment, a G-spacer is positioned between, e.g., atherapeutic element and a translocation element, a therapeutic elementand a binding element, a therapeutic element and an exogenous proteasecleavage site. In other aspects of this embodiment, an A-spacer ispositioned between, e.g., a therapeutic element and a translocationelement, a therapeutic element and a binding element, a therapeuticelement and an exogenous protease cleavage site.

In other aspects of this embodiment, a flexible spacer is positionedbetween, e.g., a binding element and a translocation element, a bindingelement and a therapeutic element, a binding element and an exogenousprotease cleavage site. In other aspects of this embodiment, a G-spaceris positioned between, e.g., a binding element and a translocationelement, a binding element and a therapeutic element, a binding elementand an exogenous protease cleavage site. In other aspects of thisembodiment, an A-spacer is positioned between, e.g., a binding elementand a translocation element, a binding element and a therapeuticelement, a binding element and an exogenous protease cleavage site.

In yet other aspects of this embodiment, a flexible spacer is positionedbetween, e.g., a translocation element and a therapeutic element, atranslocation element and a binding element, a translocation element andan exogenous protease cleavage site. In other aspects of thisembodiment, a G-spacer is positioned between, e.g., a translocationelement and a therapeutic element, a translocation element and a bindingelement, a translocation element and an exogenous protease cleavagesite. In other aspects of this embodiment, an A-spacer is positionedbetween, e.g., a translocation element and a therapeutic element, atranslocation element and a binding element, a translocation element andan exogenous protease cleavage site.

In another aspect, the invention is drawn to recombinant single-chainmodified clostridial neurotoxins that may be cleaved at will by aprotease to provide an active di-chain molecule. Such modifiedneurotoxins need not be toxic; in certain of these proteins theenzymatic activity of the toxin L chain may be abrogated, and the toxinjoined to a drug or other bioactive agent having therapeutic activity.Alternatively, in certain other modified neurotoxins the L chain isenzymatically active, but portions of the H chain are modified toprovide specificity to target cells other than the natural target of theneurotoxin, while maintaining the translocation andendocytosis-stimulating activities of the native toxin. Modifiedneurotoxins such as those described in this aspect of the invention aredisclosed in, for example, Dolly et al., Modified Clostridial Toxins forUse as Transport Proteins, International Patent Publication WO 95/32738(Dec. 7, 1995); Foster et al., Botulinum Toxin Derivatives Able toModify Peripheral Sensory Afferent Functions, International PatentPublication WO96/33273 (Oct. 24, 1996); Shone et al., Recombinant ToxinFragments, International Patent Application WO 98/07864 (98/07864); andDuggan and Chaddock, Conjugates of Galactose-Binding Lectins andClostridial Neurotoxins as Analgesics, International Patent PublicationWO 99/17806 (Apr. 15, 1999); Dolly et al., Compositions and Methods forExtending the Action of Clostridial Neurotoxin, International PatentPublication WO 99/55359 (Nov. 4, 1999); Keith A. Foster et al.,Clostridial Toxin Derivatives Able To Modify Peripheral Sensory AfferentFunctions, U.S. Pat. No. 5,989,545 (Nov. 23, 1999); Clifford C. Shone etal., Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617 (Oct. 8,2002); Conrad P. Quinn et al., Methods and Compounds for the Treatmentof Mucus Hypersecretion, U.S. Pat. No. 6,632,440 (Oct. 14, 2003); LanceE. Steward et al., Methods And Compositions For The Treatment OfPancreatitis, U.S. Pat. No. 6,843,998 (Jan. 18, 2005); Stephan Donovan,Clostridial Toxin Derivatives and Methods For Treating Pain, U.S. Pat.No. 7,138,127 (Nov. 21, 2006); Keith A. Foster et al., Inhibition ofSecretion from Non-Neural Cells, U.S. Patent Publication 2003/0180289(Sep. 25, 2003); these publications are incorporated by referenceherein. The present invention provides single-chain, cleavable versionsof these molecules and improved methods of making such molecules.

In another aspect, the invention comprises a modified clostridialneurotoxin derived from tetanus toxin (TeNT), or one or more of thebotulinum toxin (BoNT) subtypes in which the naturally-occurringinterchain loop region has been replace with a modified loop regioncomprising a different amino acid sequence conferring 1) resistance tocleavage by host proteases or autolytic action, and/or 2) lability to aselected protease. Preferably the cleavage site is highly specific forthe selected protease. The interchain loop region of certain clostridialneurotoxins, for example, BoNT/E, is naturally resistant to proteolyticcleavage in vivo. This protease resistance may reflect a secondary ortertiary structure that makes the loop more resistant to indigenousproteases than other clostridial neurotoxins. In one embodiment of thepresent invention, therefore, the inter-chain loop region of BoNT/E issubstituted for the natural loop region occurring an another BoNT havinggreater therapeutic activity or duration of action, for example BoNT/Aor /B. In another embodiment of the invention the loop region of BoNT/Eis modified to contain a proteolytic cleavage site highly specific to aselected protease prior to the subcloning. The otherwise highlyconserved BoNT/E loop region would be resistant to indigenous proteases,or those encountered within a human, but would retain the ability to beactivated by digestion with the selected protease.

Unless indicated otherwise, the following terms have the followingmeanings in this specification:

The “therapeutic element” of the present invention may comprise, withoutlimitation: active or inactive (i.e., modified) hormone receptors (suchas androgen, estrogen, retinoid, perioxysome proliferator and ecdysonereceptors etc.), and hormone-agonists and antagonists, nucleic acidscapable being of being used as replication, transcription, ortranslational templates (e.g., for expression of a protein drug havingthe desired biological activity or for synthesis of a nucleic acid drugas an antisense agent), enzymes, toxins (including apoptosis-inducing or-preventing agents), and the like.

In a preferred embodiment, the therapeutic element is a polypeptidecomprising a clostridial neurotoxin light chain or a portion thereofretaining the SNARE-protein sequence-specific endopeptidase activity ofa clostridial neurotoxin light chain. The amino acid sequences of thelight chain of botulinum neurotoxin (BoNT) subtypes A-G have beendetermined, as has the amino acid sequence of the light chains of thetetanus neurotoxin (TeNT), Baratii neurotoxin (BaNT), and butyricumneurotoxin (BuNT). Each chain contains the Zn⁺⁺-binding motifHis-Glu-Xaa-Xaa-His (SEQ ID NO: 66).

Recent studies of the BoNT/A light chain have revealed certain featuresimportant for the activity and specificity of the toxin towards itstarget substrate, SNAP-25. Thus, studies by Zhou et al. Biochemistry34:15175-15181 (1995) have indicated that when the light chain aminoacid residue His₂₂₇ is substituted with tyrosine, the resultingpolypeptide is unable to cleave SNAP-25; Kurazono et al., J. Biol. Chem.14721-14729 (1992) performed studies in the presynaptic cholinergicneurons of the buccal ganglia of Aplysia californica using recombinantBoNT/A light chain that indicated that the removal of 8 N-terminal or 32C-terminal residues did not abolish toxicity, but that removal of 10N-terminal or 57 C-terminal residues abolished toxicity in this system.Most recently, the crystal structure of the entire BoNT/A holotoxin hasbeen solved; the active site is indicated as involving the participationof His₂₂₂, Glu₂₂₃, His₂₂₆, Glu₂₆₁ and Tyr₃₆₅. Lacy et al., supra. (Theseresidues correspond to His₂₂₃, Glu₂₂₄, His₂₂₇, Glu₂₆₂ and Tyr₃₆₆ of theBoNT/A L chain of Kurazono et al., supra.) Interestingly, an alignmentof BoNT/A through E and TeNT light chains reveals that every such chaininvariably has these residues in positions analogous to BoNT/A. Kurazonoet al., supra.

The catalytic domain of BoNT/A is very specific for the C-terminus ofSNAP-25 and appears to require a minimum of 17 SNAP-25 amino acids forcleavage to occur. The catalytic site resembles a pocket; when the lightchained is linked to the heavy chain via the disulfide bond betweenCys₄₂₉ and Cys₄₅₃, the translocation domain of the heavy chain appearsto block access to the catalytic pocket until the light chain gainsentry to the cytosol. When the disulfide bond is then reduced, thecatalytic pocket is “opened” and the light chain is fully active.

The substrate specificities of the various clostridial neurotoxin lightchains other than BoNT/A are known. As described above, VAMP andsyntaxin are cleaved by BoNT/B, D, F, G and TeNT, and BoNT/C₁,respectively, while SNAP-25 is cleaved by BoNT/A E and C1. Therefore,the person of ordinary skill in the art could easily determine the toxinresidues essential in these subtypes for cleavage and substraterecognition (for example, by site-directed mutagenesis or deletion ofvarious regions of the toxin molecule followed by testing of proteolyticactivity and substrate specificity), and could therefore easily designvariants of the native neurotoxin light chain that retain or lack thesame or similar activity.

Aspects of the present invention provide, in part, a Clostridial toxinenzymatic domain. As used herein, the term “Clostridial toxin enzymaticdomain” means any Clostridial toxin polypeptide that can execute theenzymatic target modification step of the intoxication process. Thus, aClostridial toxin enzymatic domain specifically targets a Clostridialtoxin substrate and encompasses the proteolytic cleavage of aClostridial toxin substrate, such as, e.g., SNARE proteins like aSNAP-25 substrate, a VAMP substrate and a Syntaxin substrate.Non-limiting examples of a Clostridial toxin enzymatic domain include,e.g., a BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzymatic domain,a BoNT/F enzymatic domain, a BoNT/G enzymatic domain, a TeNT enzymaticdomain, a BaNT enzymatic domain, and a BuNT enzymatic domain. Othernon-limiting examples of a Clostridial toxin enzymatic domain include,e.g., amino acids 1-448 of SEQ ID NO: 1, amino acids 1-441 of SEQ ID NO:2, amino acids 1-449 of SEQ ID NO: 3, amino acids 1-445 of SEQ ID NO: 4,amino acids 1-422 of SEQ ID NO: 5, amino acids 1-439 of SEQ ID NO: 6,amino acids 1-446 of SEQ ID NO: 7, amino acids 1-457 of SEQ ID NO: 8,amino acids 1-431 of SEQ ID NO: 9, and amino acids 1-422 of SEQ ID NO:10.

A Clostridial toxin enzymatic domain includes, without limitation,naturally occurring Clostridial toxin enzymatic domain variants, suchas, e.g., Clostridial toxin enzymatic domain isoforms and Clostridialtoxin enzymatic domain subtypes; non-naturally occurring Clostridialtoxin enzymatic domain variants, such as, e.g., conservative Clostridialtoxin enzymatic domain variants, non-conservative Clostridial toxinenzymatic domain variants, Clostridial toxin enzymatic domain chimerics,active Clostridial toxin enzymatic domain fragments thereof, or anycombination thereof.

As used herein, the term “Clostridial toxin enzymatic domain variant,”whether naturally-occurring or non-naturally-occurring, means aClostridial toxin enzymatic domain that has at least one amino acidchange from the corresponding region of the disclosed referencesequences (Table 1) and can be described in percent identity to thecorresponding region of that reference sequence. Unless expresslyindicated, all Clostridial toxin enzymatic domain variants disclosed inthe present specification are capable of executing the enzymatic targetmodification step of the intoxication process. As non-limiting examples,a BoNT/A enzymatic domain variant comprising amino acids 1-448 of SEQ IDNO: 1 will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid region 1-448 of SEQ ID NO: 1; a BoNT/B enzymatic domain variantcomprising amino acids 1-441 of SEQ ID NO: 2 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1-441 of SEQID NO: 2; a BoNT/C1 enzymatic domain variant comprising amino acids1-449 of SEQ ID NO: 3 will have at least one amino acid difference, suchas, e.g., an amino acid substitution, deletion or addition, as comparedto the amino acid region 1-449 of SEQ ID NO: 3; a BoNT/D enzymaticdomain variant comprising amino acids 1-445 of SEQ ID NO: 4 will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region1-445 of SEQ ID NO: 4; a BoNT/E enzymatic domain variant comprisingamino acids 1-422 of SEQ ID NO: 5 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1-422 of SEQ ID NO: 5; aBoNT/F enzymatic domain variant comprising amino acids 1-439 of SEQ IDNO: 6 will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid region 1-439 of SEQ ID NO: 6; a BoNT/G enzymatic domain variantcomprising amino acids 1-446 of SEQ ID NO: 7 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1-446 of SEQID NO: 7; and a TeNT enzymatic domain variant comprising amino acids1-457 of SEQ ID NO: 8 will have at least one amino acid difference, suchas, e.g., an amino acid substitution, deletion or addition, as comparedto the amino acid region 1-457 of SEQ ID NO: 8.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxinenzymatic domain variants that differ somewhat in their amino acidsequence, and also in the nucleic acids encoding these proteins. Forexample, there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2,BoNT/A3 and BoNT/A4, with specific enzymatic domain subtypes showingapproximately 95% amino acid identity when compared to another BoNT/Aenzymatic domain subtype. As used herein, the term “naturally occurringClostridial toxin enzymatic domain variant” means any Clostridial toxinenzymatic domain produced by a naturally-occurring process, including,without limitation, Clostridial toxin enzymatic domain isoforms producedfrom alternatively-spliced transcripts, Clostridial toxin enzymaticdomain isoforms produced by spontaneous mutation and Clostridial toxinenzymatic domain subtypes. A naturally occurring Clostridial toxinenzymatic domain variant can function in substantially the same manneras the reference Clostridial toxin enzymatic domain on which thenaturally occurring Clostridial toxin enzymatic domain variant is based,and can be substituted for the reference Clostridial toxin enzymaticdomain in any aspect of the present invention. A naturally occurringClostridial toxin enzymatic domain variant may substitute one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, ten or more amino acids, 20or more amino acids, 30 or more amino acids, 40 or more amino acids, 50or more amino acids or 100 or more amino acids from the referenceClostridial toxin enzymatic domain on which the naturally occurringClostridial toxin enzymatic domain variant is based. A naturallyoccurring Clostridial toxin enzymatic domain variant can also substituteat least 10 contiguous amino acids, at least 15 contiguous amino acids,at least 20 contiguous amino acids, or at least 25 contiguous aminoacids from the reference Clostridial toxin enzymatic domain on which thenaturally occurring Clostridial toxin enzymatic domain variant is based,that possess at least 50% amino acid identity, 65% amino acid identity,75% amino acid identity, 85% amino acid identity or 95% amino acididentity to the reference Clostridial toxin enzymatic domain on whichthe naturally occurring Clostridial toxin enzymatic domain variant isbased.

A non-limiting examples of a naturally occurring Clostridial toxinenzymatic domain variant is a Clostridial toxin enzymatic domain isoformsuch as, e.g., a BoNT/A enzymatic domain isoform, a BoNT/B enzymaticdomain isoform, a BoNT/C1 enzymatic domain isoform, a BoNT/D enzymaticdomain isoform, a BoNT/E enzymatic domain isoform, a BoNT/F enzymaticdomain isoform, a BoNT/G enzymatic domain isoform, and a TeNT enzymaticdomain isoform. A Clostridial toxin enzymatic domain isoform canfunction in substantially the same manner as the reference Clostridialtoxin enzymatic domain on which the Clostridial toxin enzymatic domainisoform is based, and can be substituted for the reference Clostridialtoxin enzymatic domain in any aspect of the present invention.

Another non-limiting examples of a naturally occurring Clostridial toxinenzymatic domain variant is a Clostridial toxin enzymatic domain subtypesuch as, e.g., a enzymatic domain from subtype BoNT/A1, BoNT/A2, BoNT/A3and BoNT/A4; a enzymatic domain from subtype BoNT/B1, BoNT/B2, BoNT/Bbivalent and BoNT/B nonproteolytic; a enzymatic domain from subtypeBoNT/C1-1 and BoNT/C1-2; a enzymatic domain from subtype BoNT/E1,BoNT/E2 and BoNT/E3; and a enzymatic domain from subtype BoNT/F1,BoNT/F2, BoNT/F3 and BoNT/F4. A Clostridial toxin enzymatic domainsubtype can function in substantially the same manner as the referenceClostridial toxin enzymatic domain on which the Clostridial toxinenzymatic domain subtype is based, and can be substituted for thereference Clostridial toxin enzymatic domain in any aspect of thepresent invention.

As used herein, the term “non-naturally occurring Clostridial toxinenzymatic domain variant” means any Clostridial toxin enzymatic domainproduced with the aid of human manipulation, including, withoutlimitation, Clostridial toxin enzymatic domains produced by geneticengineering using random mutagenesis or rational design and Clostridialtoxin enzymatic domains produced by chemical synthesis. Non-limitingexamples of non-naturally occurring Clostridial toxin enzymatic domainvariants include, e.g., conservative Clostridial toxin enzymatic domainvariants, non-conservative Clostridial toxin enzymatic domain variants,Clostridial toxin enzymatic domain chimeric variants and activeClostridial toxin enzymatic domain fragments.

As used herein, the term “conservative Clostridial toxin enzymaticdomain variant” means a Clostridial toxin enzymatic domain that has atleast one amino acid substituted by another amino acid or an amino acidanalog that has at least one property similar to that of the originalamino acid from the reference Clostridial toxin enzymatic domainsequence (Table 1). Examples of properties include, without limitation,similar size, topography, charge, hydrophobicity, hydrophilicity,lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, aphysicochemical property, of the like, or any combination thereof. Aconservative Clostridial toxin enzymatic domain variant can function insubstantially the same manner as the reference Clostridial toxinenzymatic domain on which the conservative Clostridial toxin enzymaticdomain variant is based, and can be substituted for the referenceClostridial toxin enzymatic domain in any aspect of the presentinvention. A conservative Clostridial toxin enzymatic domain variant maysubstitute one or more amino acids, two or more amino acids, three ormore amino acids, four or more amino acids, five or more amino acids,ten or more amino acids, 20 or more amino acids, 30 or more amino acids,40 or more amino acids, 50 or more amino acids, 100 or more amino acids,200 or more amino acids, 300 or more amino acids, 400 or more aminoacids, or 500 or more amino acids from the reference Clostridial toxinenzymatic domain on which the conservative Clostridial toxin enzymaticdomain variant is based. A conservative Clostridial toxin enzymaticdomain variant can also substitute at least 10 contiguous amino acids,at least 15 contiguous amino acids, at least 20 contiguous amino acids,or at least 25 contiguous amino acids from the reference Clostridialtoxin enzymatic domain on which the conservative Clostridial toxinenzymatic domain variant is based, that possess at least 50% amino acididentity, 65% amino acid identity, 75% amino acid identity, 85% aminoacid identity or 95% amino acid identity to the reference Clostridialtoxin enzymatic domain on which the conservative Clostridial toxinenzymatic domain variant is based. Non-limiting examples of aconservative Clostridial toxin enzymatic domain variant include, e.g.,conservative BoNT/A enzymatic domain variants, conservative BoNT/Benzymatic domain variants, conservative BoNT/C1 enzymatic domainvariants, conservative BoNT/D enzymatic domain variants, conservativeBoNT/E enzymatic domain variants, conservative BoNT/F enzymatic domainvariants, conservative BoNT/G enzymatic domain variants, andconservative TeNT enzymatic domain variants.

As used herein, the term “non-conservative Clostridial toxin enzymaticdomain variant” means a Clostridial toxin enzymatic domain in which 1)at least one amino acid is deleted from the reference Clostridial toxinenzymatic domain on which the non-conservative Clostridial toxinenzymatic domain variant is based; 2) at least one amino acid added tothe reference Clostridial toxin enzymatic domain on which thenon-conservative Clostridial toxin enzymatic domain is based; or 3) atleast one amino acid is substituted by another amino acid or an aminoacid analog that does not share any property similar to that of theoriginal amino acid from the reference Clostridial toxin enzymaticdomain sequence (Table 1). A non-conservative Clostridial toxinenzymatic domain variant can function in substantially the same manneras the reference Clostridial toxin enzymatic domain on which thenon-conservative Clostridial toxin enzymatic domain variant is based,and can be substituted for the reference Clostridial toxin enzymaticdomain in any aspect of the present invention. A non-conservativeClostridial toxin enzymatic domain variant can delete one or more aminoacids, two or more amino acids, three or more amino acids, four or moreamino acids, five or more amino acids, and ten or more amino acids fromthe reference Clostridial toxin enzymatic domain on which thenon-conservative Clostridial toxin enzymatic domain variant is based. Anon-conservative Clostridial toxin enzymatic domain variant can add oneor more amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, and ten or moreamino acids to the reference Clostridial toxin enzymatic domain on whichthe non-conservative Clostridial toxin enzymatic domain variant isbased. A non-conservative Clostridial toxin enzymatic domain variant maysubstitute one or more amino acids, two or more amino acids, three ormore amino acids, four or more amino acids, five or more amino acids,ten or more amino acids, 20 or more amino acids, 30 or more amino acids,40 or more amino acids, 50 or more amino acids, 100 or more amino acids,200 or more amino acids, 300 or more amino acids, 400 or more aminoacids, or 500 or more amino acids from the reference Clostridial toxinenzymatic domain on which the non-conservative Clostridial toxinenzymatic domain variant is based. A non-conservative Clostridial toxinenzymatic domain variant can also substitute at least 10 contiguousamino acids, at least 15 contiguous amino acids, at least 20 contiguousamino acids, or at least 25 contiguous amino acids from the referenceClostridial toxin enzymatic domain on which the non-conservativeClostridial toxin enzymatic domain variant is based, that possess atleast 50% amino acid identity, 65% amino acid identity, 75% amino acididentity, 85% amino acid identity or 95% amino acid identity to thereference Clostridial toxin enzymatic domain on which thenon-conservative Clostridial toxin enzymatic domain variant is based.Non-limiting examples of a non-conservative Clostridial toxin enzymaticdomain variant include, e.g., non-conservative BoNT/A enzymatic domainvariants, non-conservative BoNT/B enzymatic domain variants,non-conservative BoNT/C1 enzymatic domain variants, non-conservativeBoNT/D enzymatic domain variants, non-conservative BoNT/E enzymaticdomain variants, non-conservative BoNT/F enzymatic domain variants,non-conservative BoNT/G enzymatic domain variants, and non-conservativeTeNT enzymatic domain variants.

As used herein, the term “Clostridial toxin enzymatic domain chimeric”means a polypeptide comprising at least a portion of a Clostridial toxinenzymatic domain and at least a portion of at least one otherpolypeptide to form a toxin enzymatic domain with at least one propertydifferent from the reference Clostridial toxin enzymatic domains ofTable 1, with the proviso that this Clostridial toxin enzymatic domainchimeric is still capable of specifically targeting the core componentsof the neurotransmitter release apparatus and thus participate inexecuting the overall cellular mechanism whereby a Clostridial toxinproteolytically cleaves a substrate. Such Clostridial toxin enzymaticdomain chimerics are described in, e.g., Lance E. Steward et al.,Leucine-based Motif and Clostridial Toxins, U.S. Patent Publication2003/0027752 (Feb. 6, 2003); Lance E. Steward et al., ClostridialNeurotoxin Compositions and Modified Clostridial Neurotoxins, U.S.Patent Publication 2003/0219462 (Nov. 27, 2003); and Lance E. Steward etal., Clostridial Neurotoxin Compositions and Modified ClostridialNeurotoxins, U.S. Patent Publication 2004/0220386 (Nov. 4, 2004), eachof which is incorporated by reference in its entirety.

As used herein, the term “active Clostridial toxin enzymatic domainfragment” means any of a variety of Clostridial toxin fragmentscomprising the enzymatic domain can be useful in aspects of the presentinvention with the proviso that these enzymatic domain fragments canspecifically target the core components of the neurotransmitter releaseapparatus and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. The enzymatic domains of Clostridial toxins are approximately420-460 amino acids in length and comprise an enzymatic domain (Table1). Research has shown that the entire length of a Clostridial toxinenzymatic domain is not necessary for the enzymatic activity of theenzymatic domain. As a non-limiting example, the first eight amino acidsof the BoNT/A enzymatic domain (residues 1-8 of SEQ ID NO: 1) are notrequired for enzymatic activity. As another non-limiting example, thefirst eight amino acids of the TeNT enzymatic domain (residues 1-8 ofSEQ ID NO: 8) are not required for enzymatic activity. Likewise, thecarboxyl-terminus of the enzymatic domain is not necessary for activity.As a non-limiting example, the last 32 amino acids of the BoNT/Aenzymatic domain (residues 417-448 of SEQ ID NO: 1) are not required forenzymatic activity. As another non-limiting example, the last 31 aminoacids of the TeNT enzymatic domain (residues 427-457 of SEQ ID NO: 8)are not required for enzymatic activity. Thus, aspects of thisembodiment can include Clostridial toxin enzymatic domains comprising anenzymatic domain having a length of, e.g., at least 350 amino acids, atleast 375 amino acids, at least 400 amino acids, at least 425 aminoacids and at least 450 amino acids. Other aspects of this embodiment caninclude Clostridial toxin enzymatic domains comprising an enzymaticdomain having a length of, e.g., at most 350 amino acids, at most 375amino acids, at most 400 amino acids, at most 425 amino acids and atmost 450 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin enzymaticdomain variants and non-naturally-occurring Clostridial toxin enzymaticdomain variants, including, without limitation, global methods, localmethods and hybrid methods, such as, e.g., segment approach methods.Protocols to determine percent identity are routine procedures withinthe scope of one skilled in the art and from the teaching herein.

Global methods align sequences from the beginning to the end of themolecule and determine the best alignment by adding up scores ofindividual residue pairs and by imposing gap penalties. Non-limitingmethods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al.,CLUSTAL W: Improving the Sensitivity of Progressive Multiple SequenceAlignment Through Sequence Weighting, Position-Specific Gap Penaltiesand Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680(1994); and iterative refinement, see, e.g., Osamu Gotoh, SignificantImprovement in Accuracy of Multiple Protein Sequence Alignments byIterative Refinement as Assessed by Reference to Structural Alignments,264(4) J. Mol. Biol. 823-838 (1996).

Local methods align sequences by identifying one or more conservedmotifs shared by all of the input sequences. Non-limiting methodsinclude, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans,Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignmentof Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbssampling, see, e.g., C. E. Lawrence et al., Detecting Subtle SequenceSignals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131)Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al.,Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics: 1428-1435 (2004).

Hybrid methods combine functional aspects of both global and localalignment methods. Non-limiting methods include, e.g.,segment-to-segment comparison, see, e.g., Burkhard Morgenstern et al.,Multiple DNA and Protein Sequence Alignment Based On Segment-To-SegmentComparison, 93(22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996);T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee: A NovelAlgorithm for Multiple Sequence Alignment, 302(1) J. Mol. Biol. 205-217(2000); MUSCLE, see, e.g., Robert C. Edgar, MUSCLE: Multiple SequenceAlignment With High Score Accuracy and High Throughput, 32(5) NucleicAcids Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amarendran RSubramanian et al., DIALIGN-T: An Improved Algorithm for Segment-BasedMultiple Sequence Alignment, 6(1) BMC Bioinformatics 66 (2005).

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin enzymatic domain. Inan aspect of this embodiment, a Clostridial toxin enzymatic domaincomprises a naturally occurring Clostridial toxin enzymatic domainvariant, such as, e.g., a Clostridial toxin enzymatic domain isoform ora Clostridial toxin enzymatic domain subtype. In another aspect of thisembodiment, a Clostridial toxin enzymatic domain comprises anon-naturally occurring Clostridial toxin enzymatic domain variant, suchas, e.g., a conservative Clostridial toxin enzymatic domain variant, anon-conservative Clostridial toxin enzymatic domain variant, aClostridial toxin chimeric enzymatic domain, an active Clostridial toxinenzymatic domain fragment, or any combination thereof.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/A enzymatic domain. In an aspect of this embodiment, a BoNT/Aenzymatic domain comprises amino acids 1-448 of SEQ ID NO: 1. In anotheraspect of this embodiment, a BoNT/A enzymatic domain comprises anaturally occurring BoNT/A enzymatic domain variant, such as, e.g., aenzymatic domain from a BoNT/A isoform or a enzymatic domain from aBoNT/A subtype. In another aspect of this embodiment, a BoNT/A enzymaticdomain comprises amino acids 1-448 of a naturally occurring BoNT/Aenzymatic domain variant of SEQ ID NO: 1, such as, e.g., amino acids1-448 of a BoNT/A isoform of SEQ ID NO: 1 or amino acids 1-448 of aBoNT/A subtype of SEQ ID NO: 1. In still another aspect of thisembodiment, a BoNT/A enzymatic domain comprises a non-naturallyoccurring BoNT/A enzymatic domain variant, such as, e.g., a conservativeBoNT/A enzymatic domain variant, a non-conservative BoNT/A enzymaticdomain variant, a BoNT/A chimeric enzymatic domain, an active BoNT/Aenzymatic domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/A enzymatic domain comprises aminoacids 1-448 of a non-naturally occurring BoNT/A enzymatic domain variantof SEQ ID NO: 1, such as, e.g., amino acids 1-448 of a conservativeBoNT/A enzymatic domain variant of SEQ ID NO: 1, amino acids 1-448 of anon-conservative BoNT/A enzymatic domain variant of SEQ ID NO: 1, aminoacids 1-448 of an active BoNT/A enzymatic domain fragment of SEQ ID NO:1, or any combination thereof.

In other aspects of this embodiment, a BoNT/A enzymatic domain comprisesa polypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 75% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 80% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 85% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 90% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1 or at least 95% amino acid identity withamino acids 1-448 of SEQ ID NO: 1. In yet other aspects of thisembodiment, a BoNT/A enzymatic domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 1-448 of SEQ IDNO: 1, at most 75% amino acid identity with amino acids 1-448 of SEQ IDNO: 1, at most 80% amino acid identity with amino acids 1-448 of SEQ IDNO: 1, at most 85% amino acid identity with amino acids 1-448 of SEQ IDNO: 1, at most 90% amino acid identity with amino acids 1-448 of SEQ IDNO: 1 or at most 95% amino acid identity with amino acids 1-448 of SEQID NO: 1.

In other aspects of this embodiment, a BoNT/A enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-448 of SEQ ID NO: 1. Inother aspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-448 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-448 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-448 of SEQ ID NO: 1. In stillother aspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-448 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-448 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-448 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-448 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-448 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-448 of SEQ ID NO: 1. In still otheraspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-448 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-448 of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/B enzymatic domain. In an aspect of this embodiment, a BoNT/Benzymatic domain comprises amino acids 1-441 of SEQ ID NO: 2. In anotheraspect of this embodiment, a BoNT/B enzymatic domain comprises anaturally occurring BoNT/B enzymatic domain variant, such as, e.g., aenzymatic domain from a BoNT/B isoform or a enzymatic domain from aBoNT/B subtype. In another aspect of this embodiment, a BoNT/B enzymaticdomain comprises amino acids 1-441 of a naturally occurring BoNT/Benzymatic domain variant of SEQ ID NO: 2, such as, e.g., amino acids1-441 of a BoNT/B isoform of SEQ ID NO: 2 or amino acids 1-441 of aBoNT/B subtype of SEQ ID NO: 2. In still another aspect of thisembodiment, a BoNT/B enzymatic domain comprises a non-naturallyoccurring BoNT/B enzymatic domain variant, such as, e.g., a conservativeBoNT/B enzymatic domain variant, a non-conservative BoNT/B enzymaticdomain variant, a BoNT/B chimeric enzymatic domain, an active BoNT/Benzymatic domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/B enzymatic domain comprises aminoacids 1-441 of a non-naturally occurring BoNT/B enzymatic domain variantof SEQ ID NO: 2, such as, e.g., amino acids 1-441 of a conservativeBoNT/B enzymatic domain variant of SEQ ID NO: 2, amino acids 1-441 of anon-conservative BoNT/B enzymatic domain variant of SEQ ID NO: 2, aminoacids 1-441 of an active BoNT/B enzymatic domain fragment of SEQ ID NO:2, or any combination thereof.

In other aspects of this embodiment, a BoNT/B enzymatic domain comprisesa polypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 75% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 80% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 85% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 90% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2 or at least 95% amino acid identity withamino acids 1-441 of SEQ ID NO: 2. In yet other aspects of thisembodiment, a BoNT/B enzymatic domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 1-441 of SEQ IDNO: 2, at most 75% amino acid identity with amino acids 1-441 of SEQ IDNO: 2, at most 80% amino acid identity with amino acids 1-441 of SEQ IDNO: 2, at most 85% amino acid identity with amino acids 1-441 of SEQ IDNO: 2, at most 90% amino acid identity with amino acids 1-441 of SEQ IDNO: 2 or at most 95% amino acid identity with amino acids 1-441 of SEQID NO: 2.

In other aspects of this embodiment, a BoNT/B enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-441 of SEQ ID NO: 2. Inother aspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-441 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-441 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-441 of SEQ ID NO: 2. In stillother aspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-441 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-441 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-441 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-441 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-441 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-441 of SEQ ID NO: 2. In still otheraspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-441 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-441 of SEQ ID NO: 2.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/C1 enzymatic domain. In an aspect of this embodiment, a BoNT/C1enzymatic domain comprises amino acids 1-449 of SEQ ID NO: 3. In anotheraspect of this embodiment, a BoNT/C1 enzymatic domain comprises anaturally occurring BoNT/C1 enzymatic domain variant, such as, e.g., aenzymatic domain from a BoNT/C1 isoform or a enzymatic domain from aBoNT/C1 subtype. In another aspect of this embodiment, a BoNT/C1enzymatic domain comprises amino acids 1-449 of a naturally occurringBoNT/C1 enzymatic domain variant of SEQ ID NO: 3, such as, e.g., aminoacids 1-449 of a BoNT/C1 isoform of SEQ ID NO: 3 or amino acids 1-449 ofa BoNT/C1 subtype of SEQ ID NO: 3. In still another aspect of thisembodiment, a BoNT/C1 enzymatic domain comprises a non-naturallyoccurring BoNT/C1 enzymatic domain variant, such as, e.g., aconservative BoNT/C1 enzymatic domain variant, a non-conservativeBoNT/C1 enzymatic domain variant, a BoNT/C1 chimeric enzymatic domain,an active BoNT/C1 enzymatic domain fragment, or any combination thereof.In still another aspect of this embodiment, a BoNT/C1 enzymatic domaincomprises amino acids 1-449 of a non-naturally occurring BoNT/C1enzymatic domain variant of SEQ ID NO: 3, such as, e.g., amino acids1-449 of a conservative BoNT/C1 enzymatic domain variant of SEQ ID NO:3, amino acids 1-449 of a non-conservative BoNT/C1 enzymatic domainvariant of SEQ ID NO: 3, amino acids 1-449 of an active BoNT/C1enzymatic domain fragment of SEQ ID NO: 3, or any combination thereof.

In other aspects of this embodiment, a BoNT/C1 enzymatic domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1-449 of SEQ ID NO: 3, at least 75% amino acid identitywith amino acids 1-449 of SEQ ID NO: 3, at least 80% amino acid identitywith amino acids 1-449 of SEQ ID NO: 3, at least 85% amino acid identitywith amino acids 1-449 of SEQ ID NO: 3, at least 90% amino acid identitywith amino acids 1-449 of SEQ ID NO: 3 or at least 95% amino acididentity with amino acids 1-449 of SEQ ID NO: 3. In yet other aspects ofthis embodiment, a BoNT/C1 enzymatic domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 1-449 ofSEQ ID NO: 3, at most 75% amino acid identity with amino acids 1-449 ofSEQ ID NO: 3, at most 80% amino acid identity with amino acids 1-449 ofSEQ ID NO: 3, at most 85% amino acid identity with amino acids 1-449 ofSEQ ID NO: 3, at most 90% amino acid identity with amino acids 1-449 ofSEQ ID NO: 3 or at most 95% amino acid identity with amino acids 1-449of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 enzymatic domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 1-449 ofSEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 enzymaticdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 1-449 ofSEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1enzymatic domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or200 non-contiguous amino acid deletions relative to amino acids 1-449 ofSEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 enzymaticdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid deletions relative to amino acids 1-449 of SEQID NO: 3. In still other aspects of this embodiment, a BoNT/C1 enzymaticdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 1-449 of SEQID NO: 3. In other aspects of this embodiment, a BoNT/C1 enzymaticdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 1-449 of SEQID NO: 3.

In other aspects of this embodiment, a BoNT/C1 enzymatic domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 1-449 of SEQ ID NO: 3.In other aspects of this embodiment, a BoNT/C1 enzymatic domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 1-449 of SEQ ID NO: 3.In yet other aspects of this embodiment, a BoNT/C1 enzymatic domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid deletions relative to amino acids 1-449 of SEQ ID NO: 3. Inother aspects of this embodiment, a BoNT/C1 enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-449 of SEQ ID NO: 3. In still otheraspects of this embodiment, a BoNT/C1 enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-449 of SEQ ID NO: 3.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/D enzymatic domain. In an aspect of this embodiment, a BoNT/Denzymatic domain comprises amino acids 1-445 of SEQ ID NO: 4. In anotheraspect of this embodiment, a BoNT/D enzymatic domain comprises anaturally occurring BoNT/D enzymatic domain variant, such as, e.g., aenzymatic domain from a BoNT/D isoform or a enzymatic domain from aBoNT/D subtype. In another aspect of this embodiment, a BoNT/D enzymaticdomain comprises amino acids 1-445 of a naturally occurring BoNT/Denzymatic domain variant of SEQ ID NO: 4, such as, e.g., amino acids1-445 of a BoNT/D isoform of SEQ ID NO: 4 or amino acids 1-445 of aBoNT/D subtype of SEQ ID NO: 4. In still another aspect of thisembodiment, a BoNT/D enzymatic domain comprises a non-naturallyoccurring BoNT/D enzymatic domain variant, such as, e.g., a conservativeBoNT/D enzymatic domain variant, a non-conservative BoNT/D enzymaticdomain variant, a BoNT/D chimeric enzymatic domain, an active BoNT/Denzymatic domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/D enzymatic domain comprises aminoacids 1-445 of a non-naturally occurring BoNT/D enzymatic domain variantof SEQ ID NO: 4, such as, e.g., amino acids 1-445 of a conservativeBoNT/D enzymatic domain variant of SEQ ID NO: 4, amino acids 1-445 of anon-conservative BoNT/D enzymatic domain variant of SEQ ID NO: 4, aminoacids 1-445 of an active BoNT/D enzymatic domain fragment of SEQ ID NO:4, or any combination thereof.

In other aspects of this embodiment, a BoNT/D enzymatic domain comprisesa polypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 75% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 80% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 85% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 90% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4 or at least 95% amino acid identity withamino acids 1-445 of SEQ ID NO: 4. In yet other aspects of thisembodiment, a BoNT/D enzymatic domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 1-445 of SEQ IDNO: 4, at most 75% amino acid identity with amino acids 1-445 of SEQ IDNO: 4, at most 80% amino acid identity with amino acids 1-445 of SEQ IDNO: 4, at most 85% amino acid identity with amino acids 1-445 of SEQ IDNO: 4, at most 90% amino acid identity with amino acids 1-445 of SEQ IDNO: 4 or at most 95% amino acid identity with amino acids 1-445 of SEQID NO: 4.

In other aspects of this embodiment, a BoNT/D enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-445 of SEQ ID NO: 4. Inother aspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-445 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-445 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-445 of SEQ ID NO: 4. In stillother aspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-445 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-445 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-445 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-445 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-445 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-445 of SEQ ID NO: 4. In still otheraspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-445 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-445 of SEQ ID NO: 4.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/E enzymatic domain. In an aspect of this embodiment, a BoNT/Eenzymatic domain comprises amino acids 1-422 of SEQ ID NO: 5. In anotheraspect of this embodiment, a BoNT/E enzymatic domain comprises anaturally occurring BoNT/E enzymatic domain variant, such as, e.g., aenzymatic domain from a BoNT/E isoform or a enzymatic domain from aBoNT/E subtype. In another aspect of this embodiment, a BoNT/E enzymaticdomain comprises amino acids 1-422 of a naturally occurring BoNT/Eenzymatic domain variant of SEQ ID NO: 5, such as, e.g., amino acids1-422 of a BoNT/E isoform of SEQ ID NO: 5 or amino acids 1-422 of aBoNT/E subtype of SEQ ID NO: 5. In still another aspect of thisembodiment, a BoNT/E enzymatic domain comprises a non-naturallyoccurring BoNT/E enzymatic domain variant, such as, e.g., a conservativeBoNT/E enzymatic domain variant, a non-conservative BoNT/E enzymaticdomain variant, a BoNT/E chimeric enzymatic domain, an active BoNT/Eenzymatic domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/E enzymatic domain comprises aminoacids 1-422 of a non-naturally occurring BoNT/E enzymatic domain variantof SEQ ID NO: 5, such as, e.g., amino acids 1-422 of a conservativeBoNT/E enzymatic domain variant of SEQ ID NO: 5, amino acids 1-422 of anon-conservative BoNT/E enzymatic domain variant of SEQ ID NO: 5, aminoacids 1-422 of an active BoNT/E enzymatic domain fragment of SEQ ID NO:5, or any combination thereof.

In other aspects of this embodiment, a BoNT/E enzymatic domain comprisesa polypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 75% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 80% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 85% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 90% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5 or at least 95% amino acid identity withamino acids 1-422 of SEQ ID NO: 5. In yet other aspects of thisembodiment, a BoNT/E enzymatic domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 1-422 of SEQ IDNO: 5, at most 75% amino acid identity with amino acids 1-422 of SEQ IDNO: 5, at most 80% amino acid identity with amino acids 1-422 of SEQ IDNO: 5, at most 85% amino acid identity with amino acids 1-422 of SEQ IDNO: 5, at most 90% amino acid identity with amino acids 1-422 of SEQ IDNO: 5 or at most 95% amino acid identity with amino acids 1-422 of SEQID NO: 5.

In other aspects of this embodiment, a BoNT/E enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-422 of SEQ ID NO: 5. Inother aspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-422 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-422 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-422 of SEQ ID NO: 5. In stillother aspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-422 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-422 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-422 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-422 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-422 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-422 of SEQ ID NO: 5. In still otheraspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-422 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-422 of SEQ ID NO: 5.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/F enzymatic domain. In an aspect of this embodiment, a BoNT/Fenzymatic domain comprises amino acids 1-439 of SEQ ID NO: 6. In anotheraspect of this embodiment, a BoNT/F enzymatic domain comprises anaturally occurring BoNT/F enzymatic domain variant, such as, e.g., aenzymatic domain from a BoNT/F isoform or a enzymatic domain from aBoNT/F subtype. In another aspect of this embodiment, a BoNT/F enzymaticdomain comprises amino acids 1-439 of a naturally occurring BoNT/Fenzymatic domain variant of SEQ ID NO: 6, such as, e.g., amino acids1-439 of a BoNT/F isoform of SEQ ID NO: 6 or amino acids 1-439 of aBoNT/F subtype of SEQ ID NO: 6. In still another aspect of thisembodiment, a BoNT/F enzymatic domain comprises a non-naturallyoccurring BoNT/F enzymatic domain variant, such as, e.g., a conservativeBoNT/F enzymatic domain variant, a non-conservative BoNT/F enzymaticdomain variant, a BoNT/F chimeric enzymatic domain, an active BoNT/Fenzymatic domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/F enzymatic domain comprises aminoacids 1-439 of a non-naturally occurring BoNT/F enzymatic domain variantof SEQ ID NO: 6, such as, e.g., amino acids 1-439 of a conservativeBoNT/F enzymatic domain variant of SEQ ID NO: 6, amino acids 1-439 of anon-conservative BoNT/F enzymatic domain variant of SEQ ID NO: 6, aminoacids 1-439 of an active BoNT/F enzymatic domain fragment of SEQ ID NO:6, or any combination thereof.

In other aspects of this embodiment, a BoNT/F enzymatic domain comprisesa polypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 75% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 80% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 85% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 90% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6 or at least 95% amino acid identity withamino acids 1-439 of SEQ ID NO: 6. In yet other aspects of thisembodiment, a BoNT/F enzymatic domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 1-439 of SEQ IDNO: 6, at most 75% amino acid identity with amino acids 1-439 of SEQ IDNO: 6, at most 80% amino acid identity with amino acids 1-439 of SEQ IDNO: 6, at most 85% amino acid identity with amino acids 1-439 of SEQ IDNO: 6, at most 90% amino acid identity with amino acids 1-439 of SEQ IDNO: 6 or at most 95% amino acid identity with amino acids 1-439 of SEQID NO: 6.

In other aspects of this embodiment, a BoNT/F enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-439 of SEQ ID NO: 6. Inother aspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-439 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-439 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-439 of SEQ ID NO: 6. In stillother aspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-439 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-439 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-439 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-439 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-439 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-439 of SEQ ID NO: 6. In still otheraspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-439 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-439 of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/G enzymatic domain. In an aspect of this embodiment, a BoNT/Genzymatic domain comprises amino acids 1-446 of SEQ ID NO: 7. In anotheraspect of this embodiment, a BoNT/G enzymatic domain comprises anaturally occurring BoNT/G enzymatic domain variant, such as, e.g., aenzymatic domain from a BoNT/G isoform or a enzymatic domain from aBoNT/G subtype. In another aspect of this embodiment, a BoNT/G enzymaticdomain comprises amino acids 1-446 of a naturally occurring BoNT/Genzymatic domain variant of SEQ ID NO: 7, such as, e.g., amino acids1-446 of a BoNT/G isoform of SEQ ID NO: 7 or amino acids 1-446 of aBoNT/G subtype of SEQ ID NO: 7. In still another aspect of thisembodiment, a BoNT/G enzymatic domain comprises a non-naturallyoccurring BoNT/G enzymatic domain variant, such as, e.g., a conservativeBoNT/G enzymatic domain variant, a non-conservative BoNT/G enzymaticdomain variant, a BoNT/G chimeric enzymatic domain, an active BoNT/Genzymatic domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/G enzymatic domain comprises aminoacids 1-446 of a non-naturally occurring BoNT/G enzymatic domain variantof SEQ ID NO: 7, such as, e.g., amino acids 1-446 of a conservativeBoNT/G enzymatic domain variant of SEQ ID NO: 7, amino acids 1-446 of anon-conservative BoNT/G enzymatic domain variant of SEQ ID NO: 7, aminoacids 1-446 of an active BoNT/G enzymatic domain fragment of SEQ ID NO:7, or any combination thereof.

In other aspects of this embodiment, a BoNT/G enzymatic domain comprisesa polypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 75% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 80% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 85% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 90% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7 or at least 95% amino acid identity withamino acids 1-446 of SEQ ID NO: 7. In yet other aspects of thisembodiment, a BoNT/G enzymatic domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 1-446 of SEQ IDNO: 7, at most 75% amino acid identity with amino acids 1-446 of SEQ IDNO: 7, at most 80% amino acid identity with amino acids 1-446 of SEQ IDNO: 7, at most 85% amino acid identity with amino acids 1-446 of SEQ IDNO: 7, at most 90% amino acid identity with amino acids 1-446 of SEQ IDNO: 7 or at most 95% amino acid identity with amino acids 1-446 of SEQID NO: 7.

In other aspects of this embodiment, a BoNT/G enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-446 of SEQ ID NO: 7. Inother aspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-446 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-446 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-446 of SEQ ID NO: 7. In stillother aspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-446 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-446 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-446 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-446 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-446 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-446 of SEQ ID NO: 7. In still otheraspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-446 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-446 of SEQ ID NO: 7.

In another embodiment, a Clostridial toxin enzymatic domain comprises aTeNT enzymatic domain. In an aspect of this embodiment, a TeNT enzymaticdomain comprises amino acids 1-457 of SEQ ID NO: 8. In another aspect ofthis embodiment, a TeNT enzymatic domain comprises a naturally occurringTeNT enzymatic domain variant, such as, e.g., a enzymatic domain from aTeNT isoform or a enzymatic domain from a TeNT subtype. In anotheraspect of this embodiment, a TeNT enzymatic domain comprises amino acids1-457 of a naturally occurring TeNT enzymatic domain variant of SEQ IDNO: 8, such as, e.g., amino acids 1-457 of a TeNT isoform of SEQ ID NO:8 or amino acids 1-457 of a TeNT subtype of SEQ ID NO: 8. In stillanother aspect of this embodiment, a TeNT enzymatic domain comprises anon-naturally occurring TeNT enzymatic domain variant, such as, e.g., aconservative TeNT enzymatic domain variant, a non-conservative TeNTenzymatic domain variant, a TeNT chimeric enzymatic domain, an activeTeNT enzymatic domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a TeNT enzymatic domain comprisesamino acids 1-457 of a non-naturally occurring TeNT enzymatic domainvariant of SEQ ID NO: 8, such as, e.g., amino acids 1-457 of aconservative TeNT enzymatic domain variant of SEQ ID NO: 8, amino acids1-457 of a non-conservative TeNT enzymatic domain variant of SEQ ID NO:8, amino acids 1-457 of an active TeNT enzymatic domain fragment of SEQID NO: 8, or any combination thereof.

In other aspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 75% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 80% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 85% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 90% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8 or at least 95% amino acid identity withamino acids 1-457 of SEQ ID NO: 8. In yet other aspects of thisembodiment, a TeNT enzymatic domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 1-457 of SEQ IDNO: 8, at most 75% amino acid identity with amino acids 1-457 of SEQ IDNO: 8, at most 80% amino acid identity with amino acids 1-457 of SEQ IDNO: 8, at most 85% amino acid identity with amino acids 1-457 of SEQ IDNO: 8, at most 90% amino acid identity with amino acids 1-457 of SEQ IDNO: 8 or at most 95% amino acid identity with amino acids 1-457 of SEQID NO: 8.

In other aspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-457 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-457 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-457 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-457 of SEQ ID NO: 8. In stillother aspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-457 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-457 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-457 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-457 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-457 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-457 of SEQ ID NO: 8. In still otheraspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-457 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-457 of SEQ ID NO: 8.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBaNT enzymatic domain. In an aspect of this embodiment, a BaNT enzymaticdomain comprises amino acids 1-431 of SEQ ID NO: 9. In another aspect ofthis embodiment, a BaNT enzymatic domain comprises a naturally occurringBaNT enzymatic domain variant, such as, e.g., a enzymatic domain from aBaNT isoform or a enzymatic domain from a BaNT subtype. In anotheraspect of this embodiment, a BaNT enzymatic domain comprises amino acids1-431 of a naturally occurring BaNT enzymatic domain variant of SEQ IDNO: 9, such as, e.g., amino acids 1-431 of a BaNT isoform of SEQ ID NO:9 or amino acids 1-431 of a BaNT subtype of SEQ ID NO: 9. In stillanother aspect of this embodiment, a BaNT enzymatic domain comprises anon-naturally occurring BaNT enzymatic domain variant, such as, e.g., aconservative BaNT enzymatic domain variant, a non-conservative BaNTenzymatic domain variant, a BaNT chimeric enzymatic domain, an activeBaNT enzymatic domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a BaNT enzymatic domain comprisesamino acids 1-431 of a non-naturally occurring BaNT enzymatic domainvariant of SEQ ID NO: 9, such as, e.g., amino acids 1-431 of aconservative BaNT enzymatic domain variant of SEQ ID NO: 9, amino acids1-431 of a non-conservative BaNT enzymatic domain variant of SEQ ID NO:9, amino acids 1-431 of an active BaNT enzymatic domain fragment of SEQID NO: 9, or any combination thereof.

In other aspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-431 of SEQ ID NO: 9, at least 75% amino acid identity with aminoacids 1-431 of SEQ ID NO: 9, at least 80% amino acid identity with aminoacids 1-431 of SEQ ID NO: 9, at least 85% amino acid identity with aminoacids 1-431 of SEQ ID NO: 9, at least 90% amino acid identity with aminoacids 1-431 of SEQ ID NO: 9 or at least 95% amino acid identity withamino acids 1-431 of SEQ ID NO: 9. In yet other aspects of thisembodiment, a BaNT enzymatic domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 1-431 of SEQ IDNO: 9, at most 75% amino acid identity with amino acids 1-431 of SEQ IDNO: 9, at most 80% amino acid identity with amino acids 1-431 of SEQ IDNO: 9, at most 85% amino acid identity with amino acids 1-431 of SEQ IDNO: 9, at most 90% amino acid identity with amino acids 1-431 of SEQ IDNO: 9 or at most 95% amino acid identity with amino acids 1-431 of SEQID NO: 9.

In other aspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-431 of SEQ ID NO: 9. Inother aspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-431 of SEQ ID NO: 9. In yetother aspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-431 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-431 of SEQ ID NO: 9. In stillother aspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-431 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-431 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-431 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-431 of SEQ ID NO: 9. In yetother aspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-431 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-431 of SEQ ID NO: 9. In still otheraspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-431 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-431 of SEQ ID NO: 9.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBuNT enzymatic domain. In an aspect of this embodiment, a BuNT enzymaticdomain comprises amino acids 1-422 of SEQ ID NO: 10. In another aspectof this embodiment, a BuNT enzymatic domain comprises a naturallyoccurring BuNT enzymatic domain variant, such as, e.g., a enzymaticdomain from a BuNT isoform or a enzymatic domain from a BuNT subtype. Inanother aspect of this embodiment, a BuNT enzymatic domain comprisesamino acids 1-422 of a naturally occurring BuNT enzymatic domain variantof SEQ ID NO: 10, such as, e.g., amino acids 1-422 of a BuNT isoform ofSEQ ID NO: 10 or amino acids 1-422 of a BuNT subtype of SEQ ID NO: 10.In still another aspect of this embodiment, a BuNT enzymatic domaincomprises a non-naturally occurring BuNT enzymatic domain variant, suchas, e.g., a conservative BuNT enzymatic domain variant, anon-conservative BuNT enzymatic domain variant, a BuNT chimericenzymatic domain, an active BuNT enzymatic domain fragment, or anycombination thereof. In still another aspect of this embodiment, a BuNTenzymatic domain comprises amino acids 1-422 of a non-naturallyoccurring BuNT enzymatic domain variant of SEQ ID NO: 10, such as, e.g.,amino acids 1-422 of a conservative BuNT enzymatic domain variant of SEQID NO: 10, amino acids 1-422 of a non-conservative BuNT enzymatic domainvariant of SEQ ID NO: 10, amino acids 1-422 of an active BuNT enzymaticdomain fragment of SEQ ID NO: 10, or any combination thereof.

In other aspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-422 of SEQ ID NO: 10, at least 75% amino acid identity withamino acids 1-422 of SEQ ID NO: 10, at least 80% amino acid identitywith amino acids 1-422 of SEQ ID NO: 10, at least 85% amino acididentity with amino acids 1-422 of SEQ ID NO: 10, at least 90% aminoacid identity with amino acids 1-422 of SEQ ID NO: 10 or at least 95%amino acid identity with amino acids 1-422 of SEQ ID NO: 10. In yetother aspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 1-422 of SEQ ID NO: 10, at most 75% amino acid identity with aminoacids 1-422 of SEQ ID NO: 10, at most 80% amino acid identity with aminoacids 1-422 of SEQ ID NO: 10, at most 85% amino acid identity with aminoacids 1-422 of SEQ ID NO: 10, at most 90% amino acid identity with aminoacids 1-422 of SEQ ID NO: 10 or at most 95% amino acid identity withamino acids 1-422 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-422 of SEQ ID NO: 10. Inother aspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-422 of SEQ ID NO: 10. Inyet other aspects of this embodiment, a BuNT enzymatic domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-422 of SEQ ID NO: 10. In otheraspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-422 of SEQ ID NO: 10. In stillother aspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-422 of SEQ ID NO: 10. In otheraspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-422 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-422 of SEQ ID NO: 10. In otheraspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-422 of SEQ ID NO: 10. In yetother aspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-422 of SEQ ID NO: 10. In otheraspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-422 of SEQ ID NO: 10. In still otheraspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-422 of SEQ ID NO: 10. In otheraspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-422 of SEQ ID NO: 10.

The “translocation element” comprises a portion of a clostridialneurotoxin heavy chain having a translocation activity. By“translocation” is meant the ability to facilitate the transport of apolypeptide through a vesicular membrane, thereby exposing some or allof the polypeptide to the cytoplasm. In the various botulinumneurotoxins translocation is thought to involve an allostericconformational change of the heavy chain caused by a decrease in pHwithin the endosome. This conformational change appears to involve andbe mediated by the N terminal half of the heavy chain and to result inthe formation of pores in the vesicular membrane; this change permitsthe movement of the proteolytic light chain from within the endosomalvesicle into the cytoplasm. See e.g., Lacy, et al., Nature Struct. Biol.5:898-902 (October 1998).

The amino acid sequence of the translocation-mediating portion of thebotulinum neurotoxin heavy chain is known to those of skill in the art;additionally, those amino acid residues within this portion that areknown to be essential for conferring the translocation activity are alsoknown. It would therefore be well within the ability of one of ordinaryskill in the art, for example, to employ the naturally occurringN-terminal peptide half of the heavy chain of any of the variousClostridium tetanus or Clostridium botulinum neurotoxin subtypes as atranslocation element, or to design an analogous translocation elementby aligning the primary sequences of the N-terminal halves of thevarious heavy chains and selecting a consensus primary translocationsequence based on conserved amino acid, polarity, steric andhydrophobicity characteristics between the sequences.

Aspects of the present invention provide, in part, a Clostridial toxintranslocation domain. As used herein, the term “Clostridial toxintranslocation domain” means any Clostridial toxin polypeptide that canexecute the translocation step of the intoxication process that mediatesClostridial toxin light chain translocation. Thus, a Clostridial toxintranslocation domain facilitates the movement of a Clostridial toxinlight chain across a membrane and encompasses the movement of aClostridial toxin light chain through the membrane an intracellularvesicle into the cytoplasm of a cell. Non-limiting examples of aClostridial toxin translocation domain include, e.g., a BoNT/Atranslocation domain, a BoNT/B translocation domain, a BoNT/C1translocation domain, a BoNT/D translocation domain, a BoNT/Etranslocation domain, a BoNT/F translocation domain, a BoNT/Gtranslocation domain, a TeNT translocation domain, a BaNT translocationdomain, and a BuNT translocation domain. Other non-limiting examples ofa Clostridial toxin translocation domain include, e.g., amino acids449-873 of SEQ ID NO: 1, amino acids 442-860 of SEQ ID NO: 2, aminoacids 450-868 of SEQ ID NO: 3, amino acids 446-864 of SEQ ID NO: 4,amino acids 423-847 of SEQ ID NO: 5, amino acids 440-866 of SEQ ID NO:6, amino acids 447-865 of SEQ ID NO: 7, amino acids 458-881 of SEQ IDNO: 8, amino acids 432-857 of SEQ ID NO: 9, and amino acids 423-847 ofSEQ ID NO: 10.

A Clostridial toxin translocation domain includes, without limitation,naturally occurring Clostridial toxin translocation domain variants,such as, e.g., Clostridial toxin translocation domain isoforms andClostridial toxin translocation domain subtypes; non-naturally occurringClostridial toxin translocation domain variants, such as, e.g.,conservative Clostridial toxin translocation domain variants,non-conservative Clostridial toxin translocation domain variants,Clostridial toxin translocation domain chimerics, active Clostridialtoxin translocation domain fragments thereof, or any combinationthereof.

As used herein, the term “Clostridial toxin translocation domainvariant,” whether naturally-occurring or non-naturally-occurring, meansa Clostridial toxin translocation domain that has at least one aminoacid change from the corresponding region of the disclosed referencesequences (Table 1) and can be described in percent identity to thecorresponding region of that reference sequence. Unless expresslyindicated, all Clostridial toxin translocation domain variants disclosedin the present specification are capable of executing the translocationstep of the intoxication process that mediates Clostridial toxin lightchain translocation. As non-limiting examples, a BoNT/A translocationdomain variant comprising amino acids 449-873 of SEQ ID NO: 1 will haveat least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region449-873 of SEQ ID NO: 1; a BoNT/B translocation domain variantcomprising amino acids 442-860 of SEQ ID NO: 2 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 442-860 ofSEQ ID NO: 2; a BoNT/C1 translocation domain variant comprising aminoacids 450-868 of SEQ ID NO: 3 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 450-868 of SEQ ID NO: 3;a BoNT/D translocation domain variant comprising amino acids 446-864 ofSEQ ID NO: 4 will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared tothe amino acid region 446-864 of SEQ ID NO: 4; a BoNT/E translocationdomain variant comprising amino acids 423-847 of SEQ ID NO: 5 will haveat least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region423-847 of SEQ ID NO: 5; a BoNT/F translocation domain variantcomprising amino acids 440-866 of SEQ ID NO: 6 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 440-866 ofSEQ ID NO: 6; a BoNT/G translocation domain variant comprising aminoacids 447-865 of SEQ ID NO: 7 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 447-865 of SEQ ID NO: 7;a TeNT translocation domain variant comprising amino acids 458-881 ofSEQ ID NO: 8 will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared tothe amino acid region 458-881 of SEQ ID NO: 8; a BaNT translocationdomain variant comprising amino acids 432-857 of SEQ ID NO: 9 will haveat least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region432-857 of SEQ ID NO: 9; and a BuNT translocation domain variantcomprising amino acids 423-847 of SEQ ID NO: 10 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 423-847 ofSEQ ID NO: 10.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxintranslocation domain variants that differ somewhat in their amino acidsequence, and also in the nucleic acids encoding these proteins. Forexample, there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2,BoNT/A3 and BoNT/A4, with specific translocation domain subtypes showingapproximately 87% amino acid identity when compared to another BoNT/Atranslocation domain subtype. As used herein, the term “naturallyoccurring Clostridial toxin translocation domain variant” means anyClostridial toxin translocation domain produced by a naturally-occurringprocess, including, without limitation, Clostridial toxin translocationdomain isoforms produced from alternatively-spliced transcripts,Clostridial toxin translocation domain isoforms produced by spontaneousmutation and Clostridial toxin translocation domain subtypes. Anaturally occurring Clostridial toxin translocation domain variant canfunction in substantially the same manner as the reference Clostridialtoxin translocation domain on which the naturally occurring Clostridialtoxin translocation domain variant is based, and can be substituted forthe reference Clostridial toxin translocation domain in any aspect ofthe present invention. A naturally occurring Clostridial toxintranslocation domain variant may substitute one or more amino acids, twoor more amino acids, three or more amino acids, four or more aminoacids, five or more amino acids, ten or more amino acids, 20 or moreamino acids, 30 or more amino acids, 40 or more amino acids, 50 or moreamino acids or 100 or more amino acids from the reference Clostridialtoxin translocation domain on which the naturally occurring Clostridialtoxin translocation domain variant is based. A naturally occurringClostridial toxin translocation domain variant can also substitute atleast 10 contiguous amino acids, at least 15 contiguous amino acids, atleast 20 contiguous amino acids, or at least 25 contiguous amino acidsfrom the reference Clostridial toxin translocation domain on which thenaturally occurring Clostridial toxin translocation domain variant isbased, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference Clostridial toxin translocation domain onwhich the naturally occurring Clostridial toxin translocation domainvariant is based.

A non-limiting examples of a naturally occurring Clostridial toxintranslocation domain variant is a Clostridial toxin translocation domainisoform such as, e.g., a BoNT/A translocation domain isoform, a BoNT/Btranslocation domain isoform, a BoNT/C1 translocation domain isoform, aBoNT/D translocation domain isoform, a BoNT/E translocation domainisoform, a BoNT/F translocation domain isoform, a BoNT/G translocationdomain isoform, a TeNT translocation domain isoform, a BaNTtranslocation domain isoform, and a BuNT translocation domain isoform. AClostridial toxin translocation domain isoform can function insubstantially the same manner as the reference Clostridial toxintranslocation domain on which the Clostridial toxin translocation domainisoform is based, and can be substituted for the reference Clostridialtoxin translocation domain in any aspect of the present invention.

Another non-limiting examples of a naturally occurring Clostridial toxintranslocation domain variant is a Clostridial toxin translocation domainsubtype such as, e.g., a translocation domain from subtype BoNT/A1,BoNT/A2, BoNT/A3 and BoNT/A4; a translocation domain from subtypeBoNT/B1, BoNT/B2, BoNT/B bivalent and BoNT/B nonproteolytic; atranslocation domain from subtype BoNT/C1-1 and BoNT/C1-2; atranslocation domain from subtype BoNT/E1, BoNT/E2 and BoNT/E3; and atranslocation domain from subtype BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4.A Clostridial toxin translocation domain subtype can function insubstantially the same manner as the reference Clostridial toxintranslocation domain on which the Clostridial toxin translocation domainsubtype is based, and can be substituted for the reference Clostridialtoxin translocation domain in any aspect of the present invention.

As used herein, the term “non-naturally occurring Clostridial toxintranslocation domain variant” means any Clostridial toxin translocationdomain produced with the aid of human manipulation, including, withoutlimitation, Clostridial toxin translocation domains produced by geneticengineering using random mutagenesis or rational design and Clostridialtoxin translocation domains produced by chemical synthesis. Non-limitingexamples of non-naturally occurring Clostridial toxin translocationdomain variants include, e.g., conservative Clostridial toxintranslocation domain variants, non-conservative Clostridial toxintranslocation domain variants, Clostridial toxin translocation domainchimeric variants and active Clostridial toxin translocation domainfragments.

As used herein, the term “conservative Clostridial toxin translocationdomain variant” means a Clostridial toxin translocation domain that hasat least one amino acid substituted by another amino acid or an aminoacid analog that has at least one property similar to that of theoriginal amino acid from the reference Clostridial toxin translocationdomain sequence (Table 1). Examples of properties include, withoutlimitation, similar size, topography, charge, hydrophobicity,hydrophilicity, lipophilicity, covalent-bonding capacity,hydrogen-bonding capacity, a physicochemical property, of the like, orany combination thereof. A conservative Clostridial toxin translocationdomain variant can function in substantially the same manner as thereference Clostridial toxin translocation domain on which theconservative Clostridial toxin translocation domain variant is based,and can be substituted for the reference Clostridial toxin translocationdomain in any aspect of the present invention. A conservativeClostridial toxin translocation domain variant may substitute one ormore amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, ten or more aminoacids, 20 or more amino acids, 30 or more amino acids, 40 or more aminoacids, 50 or more amino acids, 100 or more amino acids, 200 or moreamino acids, 300 or more amino acids, 400 or more amino acids, or 500 ormore amino acids from the reference Clostridial toxin translocationdomain on which the conservative Clostridial toxin translocation domainvariant is based. A conservative Clostridial toxin translocation domainvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference Clostridial toxintranslocation domain on which the conservative Clostridial toxintranslocation domain variant is based, that possess at least 50% aminoacid identity, 65% amino acid identity, 75% amino acid identity, 85%amino acid identity or 95% amino acid identity to the referenceClostridial toxin translocation domain on which the conservativeClostridial toxin translocation domain variant is based. Non-limitingexamples of a conservative Clostridial toxin translocation domainvariant include, e.g., conservative BoNT/A translocation domainvariants, conservative BoNT/B translocation domain variants,conservative BoNT/C1 translocation domain variants, conservative BoNT/Dtranslocation domain variants, conservative BoNT/E translocation domainvariants, conservative BoNT/F translocation domain variants,conservative BoNT/G translocation domain variants, conservative TeNTtranslocation domain variants, conservative BaNT translocation domainvariants, and conservative BuNT translocation domain variants.

As used herein, the term “non-conservative Clostridial toxintranslocation domain variant” means a Clostridial toxin translocationdomain in which 1) at least one amino acid is deleted from the referenceClostridial toxin translocation domain on which the non-conservativeClostridial toxin translocation domain variant is based; 2) at least oneamino acid added to the reference Clostridial toxin translocation domainon which the non-conservative Clostridial toxin translocation domain isbased; or 3) at least one amino acid is substituted by another aminoacid or an amino acid analog that does not share any property similar tothat of the original amino acid from the reference Clostridial toxintranslocation domain sequence (Table 1). A non-conservative Clostridialtoxin translocation domain variant can function in substantially thesame manner as the reference Clostridial toxin translocation domain onwhich the non-conservative Clostridial toxin translocation domainvariant is based, and can be substituted for the reference Clostridialtoxin translocation domain in any aspect of the present invention. Anon-conservative Clostridial toxin translocation domain variant candelete one or more amino acids, two or more amino acids, three or moreamino acids, four or more amino acids, five or more amino acids, and tenor more amino acids from the reference Clostridial toxin translocationdomain on which the non-conservative Clostridial toxin translocationdomain variant is based. A non-conservative Clostridial toxintranslocation domain variant can add one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids,five or more amino acids, and ten or more amino acids to the referenceClostridial toxin translocation domain on which the non-conservativeClostridial toxin translocation domain variant is based. Anon-conservative Clostridial toxin translocation domain variant maysubstitute one or more amino acids, two or more amino acids, three ormore amino acids, four or more amino acids, five or more amino acids,ten or more amino acids, 20 or more amino acids, 30 or more amino acids,40 or more amino acids, 50 or more amino acids, 100 or more amino acids,200 or more amino acids, 300 or more amino acids, 400 or more aminoacids, or 500 or more amino acids from the reference Clostridial toxintranslocation domain on which the non-conservative Clostridial toxintranslocation domain variant is based. A non-conservative Clostridialtoxin translocation domain variant can also substitute at least 10contiguous amino acids, at least 15 contiguous amino acids, at least 20contiguous amino acids, or at least 25 contiguous amino acids from thereference Clostridial toxin translocation domain on which thenon-conservative Clostridial toxin translocation domain variant isbased, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference Clostridial toxin translocation domain onwhich the non-conservative Clostridial toxin translocation domainvariant is based. Non-limiting examples of a non-conservativeClostridial toxin translocation domain variant include, e.g.,non-conservative BoNT/A translocation domain variants, non-conservativeBoNT/B translocation domain variants, non-conservative BoNT/C1translocation domain variants, non-conservative BoNT/D translocationdomain variants, non-conservative BoNT/E translocation domain variants,non-conservative BoNT/F translocation domain variants, non-conservativeBoNT/G translocation domain variants, and non-conservative TeNTtranslocation domain variants, non-conservative BaNT translocationdomain variants, and non-conservative BuNT translocation domainvariants.

As used herein, the term “Clostridial toxin translocation domainchimeric” means a polypeptide comprising at least a portion of aClostridial toxin translocation domain and at least a portion of atleast one other polypeptide to form a toxin translocation domain with atleast one property different from the reference Clostridial toxintranslocation domains of Table 1, with the proviso that this Clostridialtoxin translocation domain chimeric is still capable of specificallytargeting the core components of the neurotransmitter release apparatusand thus participate in executing the overall cellular mechanism wherebya Clostridial toxin proteolytically cleaves a substrate.

As used herein, the term “active Clostridial toxin translocation domainfragment” means any of a variety of Clostridial toxin fragmentscomprising the translocation domain can be useful in aspects of thepresent invention with the proviso that these active fragments canfacilitate the release of the LC from intracellular vesicles into thecytoplasm of the target cell and thus participate in executing theoverall cellular mechanism whereby a Clostridial toxin proteolyticallycleaves a substrate. The translocation domains from the heavy chains ofClostridial toxins are approximately 410-430 amino acids in length andcomprise a translocation domain (Table 1). Research has shown that theentire length of a translocation domain from a Clostridial toxin heavychain is not necessary for the translocating activity of thetranslocation domain. Thus, aspects of this embodiment can includeClostridial toxin translocation domains comprising a translocationdomain having a length of, e.g., at least 350 amino acids, at least 375amino acids, at least 400 amino acids and at least 425 amino acids.Other aspects of this embodiment can include Clostridial toxintranslocation domains comprising translocation domain having a lengthof, e.g., at most 350 amino acids, at most 375 amino acids, at most 400amino acids and at most 425 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin translocationdomain variants and non-naturally-occurring Clostridial toxintranslocation domain variants, including, without limitation, globalmethods, local methods and hybrid methods, such as, e.g., segmentapproach methods. Protocols to determine percent identity are routineprocedures within the scope of one skilled in the art and from theteaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin translocationdomain. In an aspect of this embodiment, a Clostridial toxintranslocation domain comprises a naturally occurring Clostridial toxintranslocation domain variant, such as, e.g., a Clostridial toxintranslocation domain isoform or a Clostridial toxin translocation domainsubtype. In another aspect of this embodiment, a Clostridial toxintranslocation domain comprises a non-naturally occurring Clostridialtoxin translocation domain variant, such as, e.g., a conservativeClostridial toxin translocation domain variant, a non-conservativeClostridial toxin translocation domain variant, a Clostridial toxinchimeric translocation domain, an active Clostridial toxin translocationdomain fragment, or any combination thereof.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/A translocation domain. In an aspect of thisembodiment, a BoNT/A translocation domain comprises amino acids 449-873of SEQ ID NO: 1. In another aspect of this embodiment, a BoNT/Atranslocation domain comprises a naturally occurring BoNT/Atranslocation domain variant, such as, e.g., a translocation domain froma BoNT/A isoform or a translocation domain from a BoNT/A subtype. Inanother aspect of this embodiment, a BoNT/A translocation domaincomprises amino acids 449-873 of a naturally occurring BoNT/Atranslocation domain variant of SEQ ID NO: 1, such as, e.g., amino acids449-873 of a BoNT/A isoform of SEQ ID NO: 1 or amino acids 449-873 of aBoNT/A subtype of SEQ ID NO: 1. In still another aspect of thisembodiment, a BoNT/A translocation domain comprises a non-naturallyoccurring BoNT/A translocation domain variant, such as, e.g., aconservative BoNT/A translocation domain variant, a non-conservativeBoNT/A translocation domain variant, a BoNT/A chimeric translocationdomain, an active BoNT/A translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/A translocation domain comprises amino acids 449-873 of anon-naturally occurring BoNT/A translocation domain variant of SEQ IDNO: 1, such as, e.g., amino acids 449-873 of a conservative BoNT/Atranslocation domain variant of SEQ ID NO: 1, amino acids 449-873 of anon-conservative BoNT/A translocation domain variant of SEQ ID NO: 1,amino acids 449-873 of an active BoNT/A translocation domain fragment ofSEQ ID NO: 1, or any combination thereof.

In other aspects of this embodiment, a BoNT/A translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 449-873 of SEQ ID NO: 1, at least 75% amino acididentity with amino acids 449-873 of SEQ ID NO: 1, at least 80% aminoacid identity with amino acids 449-873 of SEQ ID NO: 1, at least 85%amino acid identity with amino acids 449-873 of SEQ ID NO: 1, at least90% amino acid identity with amino acids 449-873 of SEQ ID NO: 1 or atleast 95% amino acid identity with amino acids 449-873 of SEQ ID NO: 1.In yet other aspects of this embodiment, a BoNT/A translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 449-873 of SEQ ID NO: 1, at most 75% amino acididentity with amino acids 449-873 of SEQ ID NO: 1, at most 80% aminoacid identity with amino acids 449-873 of SEQ ID NO: 1, at most 85%amino acid identity with amino acids 449-873 of SEQ ID NO: 1, at most90% amino acid identity with amino acids 449-873 of SEQ ID NO: 1 or atmost 95% amino acid identity with amino acids 449-873 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 449-873of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/Atranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids449-873 of SEQ ID NO: 1. In yet other aspects of this embodiment, aBoNT/A translocation domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 449-873 of SEQ ID NO: 1. In other aspects of this embodiment, aBoNT/A translocation domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 449-873 of SEQ ID NO: 1. In still other aspects of thisembodiment, a BoNT/A translocation domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 449-873 of SEQ ID NO: 1. In other aspects ofthis embodiment, a BoNT/A translocation domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 449-873 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 449-873 of SEQ IDNO: 1. In other aspects of this embodiment, a BoNT/A translocationdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid substitutions relative to amino acids 449-873 ofSEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/Atranslocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 449-873of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/Atranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 449-873of SEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/Atranslocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 449-873of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/Atranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 449-873of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/B translocation domain. In an aspect of thisembodiment, a BoNT/B translocation domain comprises amino acids 442-860of SEQ ID NO: 2. In another aspect of this embodiment, a BoNT/Btranslocation domain comprises a naturally occurring BoNT/Btranslocation domain variant, such as, e.g., a translocation domain froma BoNT/B isoform or a translocation domain from a BoNT/B subtype. Inanother aspect of this embodiment, a BoNT/B translocation domaincomprises amino acids 442-860 of a naturally occurring BoNT/Btranslocation domain variant of SEQ ID NO: 2, such as, e.g., amino acids442-860 of a BoNT/B isoform of SEQ ID NO: 2 or amino acids 442-860 of aBoNT/B subtype of SEQ ID NO: 2. In still another aspect of thisembodiment, a BoNT/B translocation domain comprises a non-naturallyoccurring BoNT/B translocation domain variant, such as, e.g., aconservative BoNT/B translocation domain variant, a non-conservativeBoNT/B translocation domain variant, a BoNT/B chimeric translocationdomain, an active BoNT/B translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/B translocation domain comprises amino acids 442-860 of anon-naturally occurring BoNT/B translocation domain variant of SEQ IDNO: 2, such as, e.g., amino acids 442-860 of a conservative BoNT/Btranslocation domain variant of SEQ ID NO: 2, amino acids 442-860 of anon-conservative BoNT/B translocation domain variant of SEQ ID NO: 2,amino acids 442-860 of an active BoNT/B translocation domain fragment ofSEQ ID NO: 2, or any combination thereof.

In other aspects of this embodiment, a BoNT/B translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 442-860 of SEQ ID NO: 2, at least 75% amino acididentity with amino acids 442-860 of SEQ ID NO: 2, at least 80% aminoacid identity with amino acids 442-860 of SEQ ID NO: 2, at least 85%amino acid identity with amino acids 442-860 of SEQ ID NO: 2, at least90% amino acid identity with amino acids 442-860 of SEQ ID NO: 2 or atleast 95% amino acid identity with amino acids 442-860 of SEQ ID NO: 2.In yet other aspects of this embodiment, a BoNT/B translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 442-860 of SEQ ID NO: 2, at most 75% amino acididentity with amino acids 442-860 of SEQ ID NO: 2, at most 80% aminoacid identity with amino acids 442-860 of SEQ ID NO: 2, at most 85%amino acid identity with amino acids 442-860 of SEQ ID NO: 2, at most90% amino acid identity with amino acids 442-860 of SEQ ID NO: 2 or atmost 95% amino acid identity with amino acids 442-860 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 442-860of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/Btranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids442-860 of SEQ ID NO: 2. In yet other aspects of this embodiment, aBoNT/B translocation domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 442-860 of SEQ ID NO: 2. In other aspects of this embodiment, aBoNT/B translocation domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 442-860 of SEQ ID NO: 2. In still other aspects of thisembodiment, a BoNT/B translocation domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 442-860 of SEQ ID NO: 2. In other aspects ofthis embodiment, a BoNT/B translocation domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 442-860 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 442-860 of SEQ ID NO:2. In other aspects of this embodiment, a BoNT/B translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 442-860 of SEQ ID NO:2. In yet other aspects of this embodiment, a BoNT/B translocationdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 442-860 of SEQID NO: 2. In other aspects of this embodiment, a BoNT/B translocationdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 442-860 of SEQID NO: 2. In still other aspects of this embodiment, a BoNT/Btranslocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 442-860of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/Btranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 442-860of SEQ ID NO: 2.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/C1 translocation domain. In an aspect of thisembodiment, a BoNT/C1 translocation domain comprises amino acids 450-868of SEQ ID NO: 3. In another aspect of this embodiment, a BoNT/C1translocation domain comprises a naturally occurring BoNT/C1translocation domain variant, such as, e.g., a translocation domain froma BoNT/C1 isoform or a translocation domain from a BoNT/C1 subtype. Inanother aspect of this embodiment, a BoNT/C1 translocation domaincomprises amino acids 450-868 of a naturally occurring BoNT/C1translocation domain variant of SEQ ID NO: 3, such as, e.g., amino acids450-868 of a BoNT/C1 isoform of SEQ ID NO: 3 or amino acids 450-868 of aBoNT/C1 subtype of SEQ ID NO: 3. In still another aspect of thisembodiment, a BoNT/C1 translocation domain comprises a non-naturallyoccurring BoNT/C1 translocation domain variant, such as, e.g., aconservative BoNT/C1 translocation domain variant, a non-conservativeBoNT/C1 translocation domain variant, a BoNT/C1 chimeric translocationdomain, an active BoNT/C1 translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/C1 translocation domain comprises amino acids 450-868 of anon-naturally occurring BoNT/C1 translocation domain variant of SEQ IDNO: 3, such as, e.g., amino acids 450-868 of a conservative BoNT/C1translocation domain variant of SEQ ID NO: 3, amino acids 450-868 of anon-conservative BoNT/C1 translocation domain variant of SEQ ID NO: 3,amino acids 450-868 of an active BoNT/C1 translocation domain fragmentof SEQ ID NO: 3, or any combination thereof.

In other aspects of this embodiment, a BoNT/C1 translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 450-868 of SEQ ID NO: 3, at least 75% amino acididentity with amino acids 450-868 of SEQ ID NO: 3, at least 80% aminoacid identity with amino acids 450-868 of SEQ ID NO: 3, at least 85%amino acid identity with amino acids 450-868 of SEQ ID NO: 3, at least90% amino acid identity with amino acids 450-868 of SEQ ID NO: 3 or atleast 95% amino acid identity with amino acids 450-868 of SEQ ID NO: 3.In yet other aspects of this embodiment, a BoNT/C1 translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 450-868 of SEQ ID NO: 3, at most 75% amino acididentity with amino acids 450-868 of SEQ ID NO: 3, at most 80% aminoacid identity with amino acids 450-868 of SEQ ID NO: 3, at most 85%amino acid identity with amino acids 450-868 of SEQ ID NO: 3, at most90% amino acid identity with amino acids 450-868 of SEQ ID NO: 3 or atmost 95% amino acid identity with amino acids 450-868 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 450-868of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1translocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids450-868 of SEQ ID NO: 3. In yet other aspects of this embodiment, aBoNT/C1 translocation domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 450-868 of SEQ ID NO: 3. In other aspects of this embodiment, aBoNT/C1 translocation domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 450-868 of SEQ ID NO: 3. In still other aspects of thisembodiment, a BoNT/C1 translocation domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 450-868 of SEQ ID NO: 3. In other aspects ofthis embodiment, a BoNT/C1 translocation domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 450-868 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 450-868 of SEQ ID NO:3. In other aspects of this embodiment, a BoNT/C1 translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 450-868 of SEQ ID NO:3. In yet other aspects of this embodiment, a BoNT/C1 translocationdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 450-868 of SEQID NO: 3. In other aspects of this embodiment, a BoNT/C1 translocationdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 450-868 of SEQID NO: 3. In still other aspects of this embodiment, a BoNT/C1translocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 450-868of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1translocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 450-868of SEQ ID NO: 3.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/D translocation domain. In an aspect of thisembodiment, a BoNT/D translocation domain comprises amino acids 446-864of SEQ ID NO: 4. In another aspect of this embodiment, a BoNT/Dtranslocation domain comprises a naturally occurring BoNT/Dtranslocation domain variant, such as, e.g., a translocation domain froma BoNT/D isoform or a translocation domain from a BoNT/D subtype. Inanother aspect of this embodiment, a BoNT/D translocation domaincomprises amino acids 446-864 of a naturally occurring BoNT/Dtranslocation domain variant of SEQ ID NO: 4, such as, e.g., amino acids446-864 of a BoNT/D isoform of SEQ ID NO: 4 or amino acids 446-864 of aBoNT/D subtype of SEQ ID NO: 4. In still another aspect of thisembodiment, a BoNT/D translocation domain comprises a non-naturallyoccurring BoNT/D translocation domain variant, such as, e.g., aconservative BoNT/D translocation domain variant, a non-conservativeBoNT/D translocation domain variant, a BoNT/D chimeric translocationdomain, an active BoNT/D translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/D translocation domain comprises amino acids 446-864 of anon-naturally occurring BoNT/D translocation domain variant of SEQ IDNO: 4, such as, e.g., amino acids 446-864 of a conservative BoNT/Dtranslocation domain variant of SEQ ID NO: 4, amino acids 446-864 of anon-conservative BoNT/D translocation domain variant of SEQ ID NO: 4,amino acids 446-864 of an active BoNT/D translocation domain fragment ofSEQ ID NO: 4, or any combination thereof.

In other aspects of this embodiment, a BoNT/D translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 446-864 of SEQ ID NO: 4, at least 75% amino acididentity with amino acids 446-864 of SEQ ID NO: 4, at least 80% aminoacid identity with amino acids 446-864 of SEQ ID NO: 4, at least 85%amino acid identity with amino acids 446-864 of SEQ ID NO: 4, at least90% amino acid identity with amino acids 446-864 of SEQ ID NO: 4 or atleast 95% amino acid identity with amino acids 446-864 of SEQ ID NO: 4.In yet other aspects of this embodiment, a BoNT/D translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 446-864 of SEQ ID NO: 4, at most 75% amino acididentity with amino acids 446-864 of SEQ ID NO: 4, at most 80% aminoacid identity with amino acids 446-864 of SEQ ID NO: 4, at most 85%amino acid identity with amino acids 446-864 of SEQ ID NO: 4, at most90% amino acid identity with amino acids 446-864 of SEQ ID NO: 4 or atmost 95% amino acid identity with amino acids 446-864 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 446-864of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/Dtranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids446-864 of SEQ ID NO: 4. In yet other aspects of this embodiment, aBoNT/D translocation domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 446-864 of SEQ ID NO: 4. In other aspects of this embodiment, aBoNT/D translocation domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 446-864 of SEQ ID NO: 4. In still other aspects of thisembodiment, a BoNT/D translocation domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 446-864 of SEQ ID NO: 4. In other aspects ofthis embodiment, a BoNT/D translocation domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 446-864 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 446-864 of SEQ ID NO:4. In other aspects of this embodiment, a BoNT/D translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 446-864 of SEQ ID NO:4. In yet other aspects of this embodiment, a BoNT/D translocationdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 446-864 of SEQID NO: 4. In other aspects of this embodiment, a BoNT/D translocationdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 446-864 of SEQID NO: 4. In still other aspects of this embodiment, a BoNT/Dtranslocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 446-864of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/Dtranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 446-864of SEQ ID NO: 4.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/E translocation domain. In an aspect of thisembodiment, a BoNT/E translocation domain comprises amino acids 423-847of SEQ ID NO: 5. In another aspect of this embodiment, a BoNT/Etranslocation domain comprises a naturally occurring BoNT/Etranslocation domain variant, such as, e.g., a translocation domain froma BoNT/E isoform or a translocation domain from a BoNT/E subtype. Inanother aspect of this embodiment, a BoNT/E translocation domaincomprises amino acids 423-847 of a naturally occurring BoNT/Etranslocation domain variant of SEQ ID NO: 5, such as, e.g., amino acids423-847 of a BoNT/E isoform of SEQ ID NO: 5 or amino acids 423-847 of aBoNT/E subtype of SEQ ID NO: 5. In still another aspect of thisembodiment, a BoNT/E translocation domain comprises a non-naturallyoccurring BoNT/E translocation domain variant, such as, e.g., aconservative BoNT/E translocation domain variant, a non-conservativeBoNT/E translocation domain variant, a BoNT/E chimeric translocationdomain, an active BoNT/E translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/E translocation domain comprises amino acids 423-847 of anon-naturally occurring BoNT/E translocation domain variant of SEQ IDNO: 5, such as, e.g., amino acids 423-847 of a conservative BoNT/Etranslocation domain variant of SEQ ID NO: 5, amino acids 423-847 of anon-conservative BoNT/E translocation domain variant of SEQ ID NO: 5,amino acids 423-847 of an active BoNT/E translocation domain fragment ofSEQ ID NO: 5, or any combination thereof.

In other aspects of this embodiment, a BoNT/E translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 423-847 of SEQ ID NO: 5, at least 75% amino acididentity with amino acids 423-847 of SEQ ID NO: 5, at least 80% aminoacid identity with amino acids 423-847 of SEQ ID NO: 5, at least 85%amino acid identity with amino acids 423-847 of SEQ ID NO: 5, at least90% amino acid identity with amino acids 423-847 of SEQ ID NO: 5 or atleast 95% amino acid identity with amino acids 423-847 of SEQ ID NO: 5.In yet other aspects of this embodiment, a BoNT/E translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 423-847 of SEQ ID NO: 5, at most 75% amino acididentity with amino acids 423-847 of SEQ ID NO: 5, at most 80% aminoacid identity with amino acids 423-847 of SEQ ID NO: 5, at most 85%amino acid identity with amino acids 423-847 of SEQ ID NO: 5, at most90% amino acid identity with amino acids 423-847 of SEQ ID NO: 5 or atmost 95% amino acid identity with amino acids 423-847 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 423-847of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/Etranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids423-847 of SEQ ID NO: 5. In yet other aspects of this embodiment, aBoNT/E translocation domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 423-847 of SEQ ID NO: 5. In other aspects of this embodiment, aBoNT/E translocation domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 423-847 of SEQ ID NO: 5. In still other aspects of thisembodiment, a BoNT/E translocation domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 423-847 of SEQ ID NO: 5. In other aspects ofthis embodiment, a BoNT/E translocation domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 423-847 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 423-847 of SEQ ID NO:5. In other aspects of this embodiment, a BoNT/E translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 423-847 of SEQ ID NO:5. In yet other aspects of this embodiment, a BoNT/E translocationdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 423-847 of SEQID NO: 5. In other aspects of this embodiment, a BoNT/E translocationdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 423-847 of SEQID NO: 5. In still other aspects of this embodiment, a BoNT/Etranslocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 423-847of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/Etranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 423-847of SEQ ID NO: 5.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/F translocation domain. In an aspect of thisembodiment, a BoNT/F translocation domain comprises amino acids 440-866of SEQ ID NO: 6. In another aspect of this embodiment, a BoNT/Ftranslocation domain comprises a naturally occurring BoNT/Ftranslocation domain variant, such as, e.g., a translocation domain froma BoNT/F isoform or a translocation domain from a BoNT/F subtype. Inanother aspect of this embodiment, a BoNT/F translocation domaincomprises amino acids 440-866 of a naturally occurring BoNT/Ftranslocation domain variant of SEQ ID NO: 6, such as, e.g., amino acids440-866 of a BoNT/F isoform of SEQ ID NO: 6 or amino acids 440-866 of aBoNT/F subtype of SEQ ID NO: 6. In still another aspect of thisembodiment, a BoNT/F translocation domain comprises a non-naturallyoccurring BoNT/F translocation domain variant, such as, e.g., aconservative BoNT/F translocation domain variant, a non-conservativeBoNT/F translocation domain variant, a BoNT/F chimeric translocationdomain, an active BoNT/F translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/F translocation domain comprises amino acids 440-866 of anon-naturally occurring BoNT/F translocation domain variant of SEQ IDNO: 6, such as, e.g., amino acids 440-866 of a conservative BoNT/Ftranslocation domain variant of SEQ ID NO: 6, amino acids 440-866 of anon-conservative BoNT/F translocation domain variant of SEQ ID NO: 6,amino acids 440-866 of an active BoNT/F translocation domain fragment ofSEQ ID NO: 6, or any combination thereof.

In other aspects of this embodiment, a BoNT/F translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 440-866 of SEQ ID NO: 6, at least 75% amino acididentity with amino acids 440-866 of SEQ ID NO: 6, at least 80% aminoacid identity with amino acids 440-866 of SEQ ID NO: 6, at least 85%amino acid identity with amino acids 440-866 of SEQ ID NO: 6, at least90% amino acid identity with amino acids 440-866 of SEQ ID NO: 6 or atleast 95% amino acid identity with amino acids 440-866 of SEQ ID NO: 6.In yet other aspects of this embodiment, a BoNT/F translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 440-866 of SEQ ID NO: 6, at most 75% amino acididentity with amino acids 440-866 of SEQ ID NO: 6, at most 80% aminoacid identity with amino acids 440-866 of SEQ ID NO: 6, at most 85%amino acid identity with amino acids 440-866 of SEQ ID NO: 6, at most90% amino acid identity with amino acids 440-866 of SEQ ID NO: 6 or atmost 95% amino acid identity with amino acids 440-866 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 440-866of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/Ftranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids440-866 of SEQ ID NO: 6. In yet other aspects of this embodiment, aBoNT/F translocation domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 440-866 of SEQ ID NO: 6. In other aspects of this embodiment, aBoNT/F translocation domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 440-866 of SEQ ID NO: 6. In still other aspects of thisembodiment, a BoNT/F translocation domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 440-866 of SEQ ID NO: 6. In other aspects ofthis embodiment, a BoNT/F translocation domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 440-866 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 440-866 of SEQ ID NO:6. In other aspects of this embodiment, a BoNT/F translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 440-866 of SEQ ID NO:6. In yet other aspects of this embodiment, a BoNT/F translocationdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 440-866 of SEQID NO: 6. In other aspects of this embodiment, a BoNT/F translocationdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 440-866 of SEQID NO: 6. In still other aspects of this embodiment, a BoNT/Ftranslocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 440-866of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/Ftranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 440-866of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/G translocation domain. In an aspect of thisembodiment, a BoNT/G translocation domain comprises amino acids 447-865of SEQ ID NO: 7. In another aspect of this embodiment, a BoNT/Gtranslocation domain comprises a naturally occurring BoNT/Gtranslocation domain variant, such as, e.g., a translocation domain froma BoNT/G isoform or a translocation domain from a BoNT/G subtype. Inanother aspect of this embodiment, a BoNT/G translocation domaincomprises amino acids 447-865 of a naturally occurring BoNT/Gtranslocation domain variant of SEQ ID NO: 7, such as, e.g., amino acids447-865 of a BoNT/G isoform of SEQ ID NO: 7 or amino acids 447-865 of aBoNT/G subtype of SEQ ID NO: 7. In still another aspect of thisembodiment, a BoNT/G translocation domain comprises a non-naturallyoccurring BoNT/G translocation domain variant, such as, e.g., aconservative BoNT/G translocation domain variant, a non-conservativeBoNT/G translocation domain variant, a BoNT/G chimeric translocationdomain, an active BoNT/G translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/G translocation domain comprises amino acids 447-865 of anon-naturally occurring BoNT/G translocation domain variant of SEQ IDNO: 7, such as, e.g., amino acids 447-865 of a conservative BoNT/Gtranslocation domain variant of SEQ ID NO: 7, amino acids 447-865 of anon-conservative BoNT/G translocation domain variant of SEQ ID NO: 7,amino acids 447-865 of an active BoNT/G translocation domain fragment ofSEQ ID NO: 7, or any combination thereof.

In other aspects of this embodiment, a BoNT/G translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 447-865 of SEQ ID NO: 7, at least 75% amino acididentity with amino acids 447-865 of SEQ ID NO: 7, at least 80% aminoacid identity with amino acids 447-865 of SEQ ID NO: 7, at least 85%amino acid identity with amino acids 447-865 of SEQ ID NO: 7, at least90% amino acid identity with amino acids 447-865 of SEQ ID NO: 7 or atleast 95% amino acid identity with amino acids 447-865 of SEQ ID NO: 7.In yet other aspects of this embodiment, a BoNT/G translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 447-865 of SEQ ID NO: 7, at most 75% amino acididentity with amino acids 447-865 of SEQ ID NO: 7, at most 80% aminoacid identity with amino acids 447-865 of SEQ ID NO: 7, at most 85%amino acid identity with amino acids 447-865 of SEQ ID NO: 7, at most90% amino acid identity with amino acids 447-865 of SEQ ID NO: 7 or atmost 95% amino acid identity with amino acids 447-865 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 447-865of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/Gtranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids447-865 of SEQ ID NO: 7. In yet other aspects of this embodiment, aBoNT/G translocation domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 447-865 of SEQ ID NO: 7. In other aspects of this embodiment, aBoNT/G translocation domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 447-865 of SEQ ID NO: 7. In still other aspects of thisembodiment, a BoNT/G translocation domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 447-865 of SEQ ID NO: 7. In other aspects ofthis embodiment, a BoNT/G translocation domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 447-865 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 447-865 of SEQ ID NO:7. In other aspects of this embodiment, a BoNT/G translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 447-865 of SEQ ID NO:7. In yet other aspects of this embodiment, a BoNT/G translocationdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 447-865 of SEQID NO: 7. In other aspects of this embodiment, a BoNT/G translocationdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 447-865 of SEQID NO: 7. In still other aspects of this embodiment, a BoNT/Gtranslocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 447-865of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/Gtranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 447-865of SEQ ID NO: 7.

In another embodiment, a Clostridial toxin translocation domaincomprises a TeNT translocation domain. In an aspect of this embodiment,a TeNT translocation domain comprises amino acids 458-881 of SEQ ID NO:8. In another aspect of this embodiment, a TeNT translocation domaincomprises a naturally occurring TeNT translocation domain variant, suchas, e.g., a translocation domain from a TeNT isoform or a translocationdomain from a TeNT subtype. In another aspect of this embodiment, a TeNTtranslocation domain comprises amino acids 458-881 of a naturallyoccurring TeNT translocation domain variant of SEQ ID NO: 8, such as,e.g., amino acids 458-881 of a TeNT isoform of SEQ ID NO: 8 or aminoacids 458-881 of a TeNT subtype of SEQ ID NO: 8. In still another aspectof this embodiment, a TeNT translocation domain comprises anon-naturally occurring TeNT translocation domain variant, such as,e.g., a conservative TeNT translocation domain variant, anon-conservative TeNT translocation domain variant, a TeNT chimerictranslocation domain, an active TeNT translocation domain fragment, orany combination thereof. In still another aspect of this embodiment, aTeNT translocation domain comprises amino acids 458-881 of anon-naturally occurring TeNT translocation domain variant of SEQ ID NO:8, such as, e.g., amino acids 458-881 of a conservative TeNTtranslocation domain variant of SEQ ID NO: 8, amino acids 458-881 of anon-conservative TeNT translocation domain variant of SEQ ID NO: 8,amino acids 458-881 of an active TeNT translocation domain fragment ofSEQ ID NO: 8, or any combination thereof.

In other aspects of this embodiment, a TeNT translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 458-881 of SEQ ID NO: 8, at least 75% amino acididentity with amino acids 458-881 of SEQ ID NO: 8, at least 80% aminoacid identity with amino acids 458-881 of SEQ ID NO: 8, at least 85%amino acid identity with amino acids 458-881 of SEQ ID NO: 8, at least90% amino acid identity with amino acids 458-881 of SEQ ID NO: 8 or atleast 95% amino acid identity with amino acids 458-881 of SEQ ID NO: 8.In yet other aspects of this embodiment, a TeNT translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 458-881 of SEQ ID NO: 8, at most 75% amino acididentity with amino acids 458-881 of SEQ ID NO: 8, at most 80% aminoacid identity with amino acids 458-881 of SEQ ID NO: 8, at most 85%amino acid identity with amino acids 458-881 of SEQ ID NO: 8, at most90% amino acid identity with amino acids 458-881 of SEQ ID NO: 8 or atmost 95% amino acid identity with amino acids 458-881 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 458-881of SEQ ID NO: 8. In other aspects of this embodiment, a TeNTtranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids458-881 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNTtranslocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid deletions relative to amino acids458-881 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNTtranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid deletions relative to amino acids458-881 of SEQ ID NO: 8. In still other aspects of this embodiment, aTeNT translocation domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 non-contiguous amino acid additions relative to aminoacids 458-881 of SEQ ID NO: 8. In other aspects of this embodiment, aTeNT translocation domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 non-contiguous amino acid additions relative to aminoacids 458-881 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 458-881 of SEQ ID NO:8. In other aspects of this embodiment, a TeNT translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 458-881 of SEQ ID NO:8. In yet other aspects of this embodiment, a TeNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid deletions relative to amino acids 458-881 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT translocation domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 458-881 of SEQ ID NO: 8. In stillother aspects of this embodiment, a TeNT translocation domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 458-881 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT translocation domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 458-881 of SEQ ID NO: 8.

In another embodiment, a Clostridial toxin translocation domaincomprises a BaNT translocation domain. In an aspect of this embodiment,a BaNT translocation domain comprises amino acids 432-857 of SEQ ID NO:9. In another aspect of this embodiment, a BaNT translocation domaincomprises a naturally occurring BaNT translocation domain variant, suchas, e.g., a translocation domain from a BaNT isoform or a translocationdomain from a BaNT subtype. In another aspect of this embodiment, a BaNTtranslocation domain comprises amino acids 432-857 of a naturallyoccurring BaNT translocation domain variant of SEQ ID NO: 9, such as,e.g., amino acids 432-857 of a BaNT isoform of SEQ ID NO: 9 or aminoacids 432-857 of a BaNT subtype of SEQ ID NO: 9. In still another aspectof this embodiment, a BaNT translocation domain comprises anon-naturally occurring BaNT translocation domain variant, such as,e.g., a conservative BaNT translocation domain variant, anon-conservative BaNT translocation domain variant, a BaNT chimerictranslocation domain, an active BaNT translocation domain fragment, orany combination thereof. In still another aspect of this embodiment, aBaNT translocation domain comprises amino acids 432-857 of anon-naturally occurring BaNT translocation domain variant of SEQ ID NO:9, such as, e.g., amino acids 432-857 of a conservative BaNTtranslocation domain variant of SEQ ID NO: 9, amino acids 432-857 of anon-conservative BaNT translocation domain variant of SEQ ID NO: 9,amino acids 432-857 of an active BaNT translocation domain fragment ofSEQ ID NO: 9, or any combination thereof.

In other aspects of this embodiment, a BaNT translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 432-857 of SEQ ID NO: 9, at least 75% amino acididentity with amino acids 432-857 of SEQ ID NO: 9, at least 80% aminoacid identity with amino acids 432-857 of SEQ ID NO: 9, at least 85%amino acid identity with amino acids 432-857 of SEQ ID NO: 9, at least90% amino acid identity with amino acids 432-857 of SEQ ID NO: 9 or atleast 95% amino acid identity with amino acids 432-857 of SEQ ID NO: 9.In yet other aspects of this embodiment, a BaNT translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 432-857 of SEQ ID NO: 9, at most 75% amino acididentity with amino acids 432-857 of SEQ ID NO: 9, at most 80% aminoacid identity with amino acids 432-857 of SEQ ID NO: 9, at most 85%amino acid identity with amino acids 432-857 of SEQ ID NO: 9, at most90% amino acid identity with amino acids 432-857 of SEQ ID NO: 9 or atmost 95% amino acid identity with amino acids 432-857 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 432-857of SEQ ID NO: 9. In other aspects of this embodiment, a BaNTtranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids432-857 of SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNTtranslocation domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid deletions relative to amino acids432-857 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNTtranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid deletions relative to amino acids432-857 of SEQ ID NO: 9. In still other aspects of this embodiment, aBaNT translocation domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 non-contiguous amino acid additions relative to aminoacids 432-857 of SEQ ID NO: 9. In other aspects of this embodiment, aBaNT translocation domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 non-contiguous amino acid additions relative to aminoacids 432-857 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 432-857 of SEQ ID NO:9. In other aspects of this embodiment, a BaNT translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 432-857 of SEQ ID NO:9. In yet other aspects of this embodiment, a BaNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid deletions relative to amino acids 432-857 of SEQ ID NO: 9. Inother aspects of this embodiment, a BaNT translocation domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 432-857 of SEQ ID NO: 9. In stillother aspects of this embodiment, a BaNT translocation domain comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 432-857 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT translocation domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 432-857 of SEQ ID NO: 9.

In another embodiment, a Clostridial toxin translocation domaincomprises a BuNT translocation domain. In an aspect of this embodiment,a BuNT translocation domain comprises amino acids 423-847 of SEQ ID NO:10. In another aspect of this embodiment, a BuNT translocation domaincomprises a naturally occurring BuNT translocation domain variant, suchas, e.g., a translocation domain from a BuNT isoform or a translocationdomain from a BuNT subtype. In another aspect of this embodiment, a BuNTtranslocation domain comprises amino acids 423-847 of a naturallyoccurring BuNT translocation domain variant of SEQ ID NO: 10, such as,e.g., amino acids 423-847 of a BuNT isoform of SEQ ID NO: 10 or aminoacids 423-847 of a BuNT subtype of SEQ ID NO: 10. In still anotheraspect of this embodiment, a BuNT translocation domain comprises anon-naturally occurring BuNT translocation domain variant, such as,e.g., a conservative BuNT translocation domain variant, anon-conservative BuNT translocation domain variant, a BuNT chimerictranslocation domain, an active BuNT translocation domain fragment, orany combination thereof. In still another aspect of this embodiment, aBuNT translocation domain comprises amino acids 423-847 of anon-naturally occurring BuNT translocation domain variant of SEQ ID NO:10, such as, e.g., amino acids 423-847 of a conservative BuNTtranslocation domain variant of SEQ ID NO: 10, amino acids 423-847 of anon-conservative BuNT translocation domain variant of SEQ ID NO: 10,amino acids 423-847 of an active BuNT translocation domain fragment ofSEQ ID NO: 10, or any combination thereof.

In other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 423-847 of SEQ ID NO: 10, at least 75% amino acididentity with amino acids 423-847 of SEQ ID NO: 10, at least 80% aminoacid identity with amino acids 423-847 of SEQ ID NO: 10, at least 85%amino acid identity with amino acids 423-847 of SEQ ID NO: 10, at least90% amino acid identity with amino acids 423-847 of SEQ ID NO: 10 or atleast 95% amino acid identity with amino acids 423-847 of SEQ ID NO: 10.In yet other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 423-847 of SEQ ID NO: 10, at most 75% amino acididentity with amino acids 423-847 of SEQ ID NO: 10, at most 80% aminoacid identity with amino acids 423-847 of SEQ ID NO: 10, at most 85%amino acid identity with amino acids 423-847 of SEQ ID NO: 10, at most90% amino acid identity with amino acids 423-847 of SEQ ID NO: 10 or atmost 95% amino acid identity with amino acids 423-847 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to amino acids 423-847of SEQ ID NO: 10. In other aspects of this embodiment, a BuNTtranslocation domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to amino acids423-847 of SEQ ID NO: 10. In yet other aspects of this embodiment, aBuNT translocation domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 423-847 of SEQ ID NO: 10. In other aspects of this embodiment, aBuNT translocation domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 423-847 of SEQ ID NO: 10. In still other aspects of thisembodiment, a BuNT translocation domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 423-847 of SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT translocation domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 423-847 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 423-847 of SEQ ID NO:10. In other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 423-847 of SEQ ID NO:10. In yet other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid deletions relative to amino acids 423-847 of SEQ ID NO: 10.In other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid deletions relative to amino acids 423-847 of SEQ ID NO: 10.In still other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 423-847 of SEQ ID NO: 10.In other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 423-847 of SEQ ID NO: 10.

By “binding element” is meant an amino acid sequence region able topreferentially bind to a cell surface marker characteristic of thetarget cell under physiological conditions. The cell surface marker maycomprise a polypeptide, a polysaccharide, a lipid, a glycoprotein, alipoprotein, or may have structural characteristics of more than one ofthese. By “preferentially interact” is meant that the disassociationconstant (K_(d)) of the binding element for the cell surface marker isat least one order of magnitude less than that of the binding elementfor any other cell surface marker. Preferably, the disassociationconstant is at least 2 orders of magnitude less, even more preferablythe disassociation constant is at least 3 orders of magnitude less thanthat of the binding element for any other cell surface marker to whichthe neurotoxin or modified neurotoxin is exposed. Examples of bindingelements are described in, e.g., Steward, L. E. et al., ModifiedClostridial Toxins with Enhanced Translocation Capability and EnhancedTargeting Activity, U.S. patent application Ser. No. 11/776,043 (Jul.11, 2007); Steward, L. E. et al., Modified Clostridial Toxins withEnhanced Translocation Capabilities and Altered Targeting Activity ForClostridial Toxin Target Cells, U.S. patent application Ser. No.11/776,052 (Jul. 11, 2007); and Steward, L. E. et al., ModifiedClostridial Toxins with Enhanced Translocation Capabilities and AlteredTargeting Activity For Non-Clostridial Toxin Target Cells, U.S. patentapplication Ser. No. 11/776,075 (Jul. 11, 2007), each of which isincorporated by reference in its entirety.

A non-limiting example of a binding element disclosed in the presentspecification is, e.g., a melanocortin peptide, such as, e.g., anα-melanocyte stimulating hormones (α-MSH), a β-melanocyte stimulatinghormones (β-MSH), a γ-melanocyte stimulating hormones (γ-MSH). Thus, inan embodiment, a binding element is derived from a melanocortin peptide.

In another embodiment, a binding element comprises a melanocytestimulating hormone. In aspects of this embodiment, a binding elementcomprises a melanocyte stimulating hormone is derived from anα-melanocyte stimulating hormones (α-MSH), a β-melanocyte stimulatinghormones (β-MSH), a γ-melanocyte stimulating hormones (γ-MSH). In otheraspects of this embodiment, a binding element comprises a melanocytestimulating hormone comprises SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO:83.

In other aspects of this embodiment, a binding element comprises amelanocyte stimulating hormone has, e.g., at least 70% amino acididentity with SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83, at least75% amino acid identity with SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO:83, at least 80% amino acid identity with SEQ ID NO: 81, SEQ ID NO: 82or SEQ ID NO: 83, at least 85% amino acid identity with SEQ ID NO: 81,SEQ ID NO: 82 or SEQ ID NO: 83, at least 90% amino acid identity withSEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83 or at least 95% amino acididentity with SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83. In yetother aspects of this embodiment, a binding element comprises amelanocyte stimulating hormone has, e.g., at most 70% amino acididentity with SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83, at most 75%amino acid identity with SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83,at most 80% amino acid identity with SEQ ID NO: 81, SEQ ID NO: 82 or SEQID NO: 83, at most 85% amino acid identity with SEQ ID NO: 81, SEQ IDNO: 82 or SEQ ID NO: 83, at most 90% amino acid identity with SEQ ID NO:81, SEQ ID NO: 82 or SEQ ID NO: 83 or at most 95% amino acid identitywith SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83.

In other aspects of this embodiment, a binding element comprises amelanocyte stimulating hormone has, e.g., at least one, two, three, fouror five non-contiguous amino acid substitutions relative to SEQ ID NO:81, SEQ ID NO: 82 or SEQ ID NO: 83. In other aspects of this embodiment,a binding element comprises a melanocyte stimulating hormone has, e.g.,at most one, two, three, four or five non-contiguous amino acidsubstitutions relative to SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83.In yet other aspects of this embodiment, a binding element comprises amelanocyte stimulating hormone has, e.g., at least one, two, three, fouror five non-contiguous amino acid deletions relative to SEQ ID NO: 81,SEQ ID NO: 82 or SEQ ID NO: 83. In yet other aspects of this embodiment,a binding element comprises a melanocyte stimulating hormone has, e.g.,at most one, two, three, four or five non-contiguous amino aciddeletions relative to SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83. Instill other aspects of this embodiment, a binding element comprises amelanocyte stimulating hormone has, e.g., at least one, two, three, fouror five non-contiguous amino acid additions relative to SEQ ID NO: 81,SEQ ID NO: 82 or SEQ ID NO: 83. In yet other aspects of this embodiment,a binding element comprises a melanocyte stimulating hormone has, e.g.,at most one, two, three, four or five non-contiguous amino acidadditions relative to SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83.

In other aspects of this embodiment, a binding element comprises amelanocyte stimulating hormone has, e.g., at least one, two, three, fouror five contiguous amino acid substitutions relative to SEQ ID NO: 81,SEQ ID NO: 82 or SEQ ID NO: 83. In other aspects of this embodiment, abinding element comprises a melanocyte stimulating hormone has, e.g., atmost one, two, three, four or five contiguous amino acid substitutionsrelative to SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83. In yet otheraspects of this embodiment, a binding element comprises a melanocytestimulating hormone has, e.g., at least one, two, three, four or fivecontiguous amino acid deletions relative to SEQ ID NO: 81, SEQ ID NO: 82or SEQ ID NO: 83. In yet other aspects of this embodiment, a bindingelement comprises a melanocyte stimulating hormone has, e.g., at mostone, two, three, four or five contiguous amino acid deletions relativeto SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83. In still other aspectsof this embodiment, a binding element comprises a melanocyte stimulatinghormone has, e.g., at least one, two, three, four or five contiguousamino acid additions relative to SEQ ID NO: 81, SEQ ID NO: 82 or SEQ IDNO: 83. In yet other aspects of this embodiment, a binding elementcomprises a melanocyte stimulating hormone has, e.g., at most one, two,three, four or five contiguous amino acid additions relative to SEQ IDNO: 81, SEQ ID NO: 82 or SEQ ID NO: 83.

In another embodiment, a binding element comprises anadrenocorticotropin. In aspects of this embodiment, a binding elementcomprising an adrenocorticotropin is derived from an adrenocorticotropin(ACTH) or a Corticotropin-like intermediary peptide (CLIP). In otheraspects of this embodiment, a binding element comprising anadrenocorticotropin comprises SEQ ID NO: 84 or SEQ ID NO: 85.

In other aspects of this embodiment, a binding element comprising anadrenocorticotropin has, e.g., at least 70% amino acid identity with SEQID NO: 84 or SEQ ID NO: 85, at least 75% amino acid identity with SEQ IDNO: 84 or SEQ ID NO: 85, at least 80% amino acid identity with SEQ IDNO: 84 or SEQ ID NO: 85, at least 85% amino acid identity with SEQ IDNO: 84 or SEQ ID NO: 85, at least 90% amino acid identity with SEQ IDNO: 84 or SEQ ID NO: 85 or at least 95% amino acid identity with SEQ IDNO: 84 or SEQ ID NO: 85. In yet other aspects of this embodiment, abinding element comprising an adrenocorticotropin has, e.g., at most 70%amino acid identity with SEQ ID NO: 84 or SEQ ID NO: 85, at most 75%amino acid identity with SEQ ID NO: 84 or SEQ ID NO: 85, at most 80%amino acid identity with SEQ ID NO: 84 or SEQ ID NO: 85, at most 85%amino acid identity with SEQ ID NO: 84 or SEQ ID NO: 85, at most 90%amino acid identity with SEQ ID NO: 84 or SEQ ID NO: 85 or at most 95%amino acid identity with SEQ ID NO: 84 or SEQ ID NO: 85.

In other aspects of this embodiment, a binding element comprising anadrenocorticotropin has, e.g., at least one, two, three, four or fivenon-contiguous amino acid substitutions relative to SEQ ID NO: 84 or SEQID NO: 85. In other aspects of this embodiment, a binding elementcomprising an adrenocorticotropin has, e.g., at most one, two, three,four or five non-contiguous amino acid substitutions relative to SEQ IDNO: 84 or SEQ ID NO: 85. In yet other aspects of this embodiment, abinding element comprising an adrenocorticotropin has, e.g., at leastone, two, three, four or five non-contiguous amino acid deletionsrelative to SEQ ID NO: 84 or SEQ ID NO: 85. In yet other aspects of thisembodiment, a binding element comprising an adrenocorticotropin has,e.g., at most one, two, three, four or five non-contiguous amino aciddeletions relative to SEQ ID NO: 84 or SEQ ID NO: 85. In still otheraspects of this embodiment, a binding element comprising anadrenocorticotropin has, e.g., at least one, two, three, four or fivenon-contiguous amino acid additions relative to SEQ ID NO: 84 or SEQ IDNO: 85. In yet other aspects of this embodiment, a binding elementcomprising an adrenocorticotropin has, e.g., at most one, two, three,four or five non-contiguous amino acid additions relative to SEQ ID NO:84 or SEQ ID NO: 85.

In other aspects of this embodiment, a binding element comprising anadrenocorticotropin has, e.g., at least one, two, three, four or fivecontiguous amino acid substitutions relative to SEQ ID NO: 84 or SEQ IDNO: 85. In other aspects of this embodiment, a binding elementcomprising an adrenocorticotropin has, e.g., at most one, two, three,four or five contiguous amino acid substitutions relative to SEQ ID NO:84 or SEQ ID NO: 85. In yet other aspects of this embodiment, a bindingelement comprising an adrenocorticotropin has, e.g., at least one, two,three, four or five contiguous amino acid deletions relative to SEQ IDNO: 84 or SEQ ID NO: 85. In yet other aspects of this embodiment, abinding element comprising an adrenocorticotropin has, e.g., at mostone, two, three, four or five contiguous amino acid deletions relativeto SEQ ID NO: 84 or SEQ ID NO: 85. In still other aspects of thisembodiment, a binding element comprising an adrenocorticotropin has,e.g., at least one, two, three, four or five contiguous amino acidadditions relative to SEQ ID NO: 84 or SEQ ID NO: 85. In yet otheraspects of this embodiment, a binding element comprising anadrenocorticotropin has, e.g., at most one, two, three, four or fivecontiguous amino acid additions relative to SEQ ID NO: 84 or SEQ ID NO:85.

In another embodiment, a binding element comprises a lipotropin. Inaspects of this embodiment, a binding element comprising a lipotropin isderived from a β-lipotropin (β-LPH) or a γ-lipotropin (γ-LPH). In otheraspects of this embodiment, a binding element comprising a lipotropincomprises SEQ ID NO: 86 or SEQ ID NO: 87.

In other aspects of this embodiment, a binding element comprising alipotropin has, e.g., at least 70% amino acid identity with SEQ ID NO:86 or SEQ ID NO: 87, at least 75% amino acid identity with SEQ ID NO: 86or SEQ ID NO: 87, at least 80% amino acid identity with SEQ ID NO: 86 orSEQ ID NO: 87, at least 85% amino acid identity with SEQ ID NO: 86 orSEQ ID NO: 87, at least 90% amino acid identity with SEQ ID NO: 86 orSEQ ID NO: 87 or at least 95% amino acid identity with SEQ ID NO: 86 orSEQ ID NO: 87. In yet other aspects of this embodiment, a bindingelement comprising a lipotropin has, e.g., at most 70% amino acididentity with SEQ ID NO: 86 or SEQ ID NO: 87, at most 75% amino acididentity with SEQ ID NO: 86 or SEQ ID NO: 87, at most 80% amino acididentity with SEQ ID NO: 86 or SEQ ID NO: 87, at most 85% amino acididentity with SEQ ID NO: 86 or SEQ ID NO: 87, at most 90% amino acididentity with SEQ ID NO: 86 or SEQ ID NO: 87 or at most 95% amino acididentity with SEQ ID NO: 86 or SEQ ID NO: 87.

In other aspects of this embodiment, a binding element comprising alipotropin has, e.g., at least one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid substitutions relative toSEQ ID NO: 86 or SEQ ID NO: 87. In other aspects of this embodiment, abinding element comprising a lipotropin has, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid substitutions relative to SEQ ID NO: 86 or SEQ ID NO: 87. In yetother aspects of this embodiment, a binding element comprising alipotropin has, e.g., at least one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid deletions relative to SEQID NO: 86 or SEQ ID NO: 87. In yet other aspects of this embodiment, abinding element comprising a lipotropin has, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid deletions relative to SEQ ID NO: 86 or SEQ ID NO: 87. In stillother aspects of this embodiment, a binding element comprising alipotropin has, e.g., at least one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid additions relative to SEQID NO: 86 or SEQ ID NO: 87. In yet other aspects of this embodiment, abinding element comprising a lipotropin has, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid additions relative to SEQ ID NO: 86 or SEQ ID NO: 87.

In other aspects of this embodiment, a binding element comprising alipotropin has, e.g., at least one, two, three, four, five, six, seven,eight, nine or ten contiguous amino acid substitutions relative to SEQID NO: 86 or SEQ ID NO: 87. In other aspects of this embodiment, abinding element comprising a lipotropin has, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 86 or SEQ ID NO: 87. In yet otheraspects of this embodiment, a binding element comprising a lipotropinhas, e.g., at least one, two, three, four, five, six, seven, eight, nineor ten contiguous amino acid deletions relative to SEQ ID NO: 86 or SEQID NO: 87. In yet other aspects of this embodiment, a binding elementcomprising a lipotropin has, e.g., at most one, two, three, four, five,six, seven, eight, nine or ten contiguous amino acid deletions relativeto SEQ ID NO: 86 or SEQ ID NO: 87. In still other aspects of thisembodiment, a binding element comprising a lipotropin has, e.g., atleast one, two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 86 or SEQ ID NO:87. In yet other aspects of this embodiment, a binding elementcomprising a lipotropin has, e.g., at most one, two, three, four, five,six, seven, eight, nine or ten contiguous amino acid additions relativeto SEQ ID NO: 86 or SEQ ID NO: 87.

In another embodiment, a binding element comprising a neuropeptidederived from a melanocortin peptide. In aspects of this embodiment, abinding element comprising a neuropeptide derived from a melanocortinpeptide comprises SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90.

In other aspects of this embodiment, a binding element comprising aneuropeptide derived from a melanocortin peptide has, e.g., at least 70%amino acid identity with SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90,at least 75% amino acid identity with SEQ ID NO: 88, SEQ ID NO: 89 orSEQ ID NO: 90, at least 80% amino acid identity with SEQ ID NO: 88, SEQID NO: 89 or SEQ ID NO: 90, at least 85% amino acid identity with SEQ IDNO: 88, SEQ ID NO: 89 or SEQ ID NO: 90, at least 90% amino acid identitywith SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90 or at least 95% aminoacid identity with SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. In yetother aspects of this embodiment, a binding element comprising aneuropeptide derived from a melanocortin peptide has, e.g., at most 70%amino acid identity with SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90,at most 75% amino acid identity with SEQ ID NO: 88, SEQ ID NO: 89 or SEQID NO: 90, at most 80% amino acid identity with SEQ ID NO: 88, SEQ IDNO: 89 or SEQ ID NO: 90, at most 85% amino acid identity with SEQ ID NO:88, SEQ ID NO: 89 or SEQ ID NO: 90, at most 90% amino acid identity withSEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90 or at most 95% amino acididentity with SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90.

In other aspects of this embodiment, a binding element comprising aneuropeptide derived from a melanocortin peptide has, e.g., at leastone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid substitutions relative to SEQ ID NO: 88, SEQID NO: 89 or SEQ ID NO: 90. In other aspects of this embodiment, abinding element comprising a neuropeptide derived from a melanocortinpeptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid substitutions relative toSEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. In yet other aspects ofthis embodiment, a binding element comprising a neuropeptide derivedfrom a melanocortin peptide has, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acid deletionsrelative to SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. In yet otheraspects of this embodiment, a binding element comprising a neuropeptidederived from a melanocortin peptide has, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino aciddeletions relative to SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. Instill other aspects of this embodiment, a binding element comprising aneuropeptide derived from a melanocortin peptide has, e.g., at leastone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 88, SEQ IDNO: 89 or SEQ ID NO: 90. In yet other aspects of this embodiment, abinding element comprising a neuropeptide derived from a melanocortinpeptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid additions relative to SEQID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90.

In other aspects of this embodiment, a binding element comprising aneuropeptide derived from a melanocortin peptide has, e.g., at leastone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid substitutions relative to SEQ ID NO: 88, SEQ ID NO: 89 or SEQID NO: 90. In other aspects of this embodiment, a binding elementcomprising a neuropeptide derived from a melanocortin peptide has, e.g.,at most one, two, three, four, five, six, seven, eight, nine or tencontiguous amino acid substitutions relative to SEQ ID NO: 88, SEQ IDNO: 89 or SEQ ID NO: 90. In yet other aspects of this embodiment, abinding element comprising a neuropeptide derived from a melanocortinpeptide has, e.g., at least one, two, three, four, five, six, seven,eight, nine or ten contiguous amino acid deletions relative to SEQ IDNO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. In yet other aspects of thisembodiment, a binding element comprising a neuropeptide derived from amelanocortin peptide has, e.g., at most one, two, three, four, five,six, seven, eight, nine or ten contiguous amino acid deletions relativeto SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. In still other aspectsof this embodiment, a binding element comprising a neuropeptide derivedfrom a melanocortin peptide has, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten contiguous amino acid additionsrelative to SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90. In yet otheraspects of this embodiment, a binding element comprising a neuropeptidederived from a melanocortin peptide has, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90.

In another embodiment, a binding element comprises a galanin. In aspectsof this embodiment, a binding element comprising a galanin is derivedfrom a galanin or a galanin message-associated peptide (GMAP). In otheraspects of this embodiment, a binding element comprising a galanincomprises SEQ ID NO: 91 or SEQ ID NO: 92.

In other aspects of this embodiment, a binding element comprising agalanin has, e.g., at least 70% amino acid identity with SEQ ID NO: 91or SEQ ID NO: 92, at least 75% amino acid identity with SEQ ID NO: 91 orSEQ ID NO: 92, at least 80% amino acid identity with SEQ ID NO: 91 orSEQ ID NO: 92, at least 85% amino acid identity with SEQ ID NO: 91 orSEQ ID NO: 92, at least 90% amino acid identity with SEQ ID NO: 91 orSEQ ID NO: 92 or at least 95% amino acid identity with SEQ ID NO: 91 orSEQ ID NO: 92. In yet other aspects of this embodiment, a bindingelement comprising a galanin has, e.g., at most 70% amino acid identitywith SEQ ID NO: 91 or SEQ ID NO: 92, at most 75% amino acid identitywith SEQ ID NO: 91 or SEQ ID NO: 92, at most 80% amino acid identitywith SEQ ID NO: 91 or SEQ ID NO: 92, at most 85% amino acid identitywith SEQ ID NO: 91 or SEQ ID NO: 92, at most 90% amino acid identitywith SEQ ID NO: 91 or SEQ ID NO: 92 or at most 95% amino acid identitywith SEQ ID NO: 91 or SEQ ID NO: 92.

In other aspects of this embodiment, a binding element comprising agalanin has, e.g., at least one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid substitutions relative toSEQ ID NO: 91 or SEQ ID NO: 92. In other aspects of this embodiment, abinding element comprising a galanin has, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 91 or SEQ ID NO: 92. In yet otheraspects of this embodiment, a binding element comprising a galanin has,e.g., at least one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid deletions relative to SEQ ID NO: 91 or SEQID NO: 92. In yet other aspects of this embodiment, a binding elementcomprising a galanin has, e.g., at most one, two, three, four, five,six, seven, eight, nine or ten non-contiguous amino acid deletionsrelative to SEQ ID NO: 91 or SEQ ID NO: 92. In still other aspects ofthis embodiment, a binding element comprising a galanin has, e.g., atleast one, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 91 or SEQ IDNO: 92. In yet other aspects of this embodiment, a binding elementcomprising a galanin has, e.g., at most one, two, three, four, five,six, seven, eight, nine or ten non-contiguous amino acid additionsrelative to SEQ ID NO: 91 or SEQ ID NO: 92.

In other aspects of this embodiment, a binding element comprising agalanin has, e.g., at least one, two, three, four, five, six, seven,eight, nine or ten contiguous amino acid substitutions relative to SEQID NO: 91 or SEQ ID NO: 92. In other aspects of this embodiment, abinding element comprising a galanin has, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 91 or SEQ ID NO: 92. In yet otheraspects of this embodiment, a binding element comprising a galanin has,e.g., at least one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid deletions relative to SEQ ID NO: 91 or SEQ IDNO: 92. In yet other aspects of this embodiment, a binding elementcomprising a galanin has, e.g., at most one, two, three, four, five,six, seven, eight, nine or ten contiguous amino acid deletions relativeto SEQ ID NO: 91 or SEQ ID NO: 92. In still other aspects of thisembodiment, a binding element comprising a galanin has, e.g., at leastone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid additions relative to SEQ ID NO: 91 or SEQ ID NO: 92. In yetother aspects of this embodiment, a binding element comprising a galaninhas, e.g., at most one, two, three, four, five, six, seven, eight, nineor ten contiguous amino acid additions relative to SEQ ID NO: 91 or SEQID NO: 92.

Another example of a binding element disclosed in the presentspecification is, e.g., a granin peptide, such as, e.g., a chromograninA, a chromogranin B (secretogranin 1) or a chromogranin C (secretograninII). Thus, in an embodiment, a binding element is derived from a graninpeptide.

In another embodiment, a binding element comprising a granin peptidecomprises a chromogranin A peptide. In aspects of this embodiment, abinding element comprising a chromogranin A peptide is derived from aβ-granin, a vasostatin, a chromostatin, a pancreastatin, a WE-14, acatestatin, a parastatin or a GE-25. In other aspects of thisembodiment, a binding element comprising a chromogranin A peptidecomprises SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96,SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100.

In other aspects of this embodiment, a binding element comprising achromogranin A peptide has, e.g., at least 70% amino acid identity withSEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100, at least 75% aminoacid identity with SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ IDNO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100,at least 80% amino acid identity with SEQ ID NO: 93, SEQ ID NO: 94, SEQID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 orSEQ ID NO: 100, at least 85% amino acid identity with SEQ ID NO: 93, SEQID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO: 99 or SEQ ID NO: 100, at least 90% amino acid identity withSEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100 or at least 95% aminoacid identity with SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ IDNO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100.In yet other aspects of this embodiment, a binding element comprising achromogranin A peptide has, e.g., at most 70% amino acid identity withSEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100, at most 75% aminoacid identity with SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ IDNO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100,at most 80% amino acid identity with SEQ ID NO: 93, SEQ ID NO: 94, SEQID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 orSEQ ID NO: 100, at most 85% amino acid identity with SEQ ID NO: 93, SEQID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO: 99 or SEQ ID NO: 100, at most 90% amino acid identity withSEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100 or at most 95% aminoacid identity with SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ IDNO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100.

In other aspects of this embodiment, a binding element comprising achromogranin A peptide has, e.g., at least one, two, three, four, five,six, seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96,SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100. In otheraspects of this embodiment, a binding element comprising a chromograninA peptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid substitutions relative toSEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100. In yet other aspectsof this embodiment, a binding element comprising a chromogranin Apeptide has, e.g., at least one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid deletions relative to SEQID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97,SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100. In yet other aspects ofthis embodiment, a binding element comprising a chromogranin A peptidehas, e.g., at most one, two, three, four, five, six, seven, eight, nineor ten non-contiguous amino acid deletions relative to SEQ ID NO: 93,SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:98, SEQ ID NO: 99 or SEQ ID NO: 100. In still other aspects of thisembodiment, a binding element comprising a chromogranin A peptide has,e.g., at least one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid additions relative to SEQ ID NO: 93, SEQID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO:99 or SEQ ID NO:100. In yet other aspects of this embodiment,a binding element comprising a chromogranin A peptide has, e.g., at mostone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 93, SEQ IDNO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQID NO: 99 or SEQ ID NO: 100.

In other aspects of this embodiment, a binding element comprising achromogranin A peptide has, e.g., at least one, two, three, four, five,six, seven, eight, nine or ten contiguous amino acid substitutionsrelative to SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96,SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100. In otheraspects of this embodiment, a binding element comprising a chromograninA peptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten contiguous amino acid substitutions relative to SEQID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97,SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100. In yet other aspects ofthis embodiment, a binding element comprising a chromogranin A peptidehas, e.g., at least one, two, three, four, five, six, seven, eight, nineor ten contiguous amino acid deletions relative to SEQ ID NO: 93, SEQ IDNO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQID NO: 99 or SEQ ID NO: 100. In yet other aspects of this embodiment, abinding element comprising a chromogranin A peptide has, e.g., at mostone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid deletions relative to SEQ ID NO: 93, SEQ ID NO: 94, SEQ IDNO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 orSEQ ID NO: 100. In still other aspects of this embodiment, a bindingelement comprising a chromogranin A peptide has, e.g., at least one,two, three, four, five, six, seven, eight, nine or ten contiguous aminoacid additions relative to SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99 or SEQ IDNO:100. In yet other aspects of this embodiment, a binding elementcomprising a chromogranin A peptide has, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO:100.

In another embodiment, a binding element comprising a chromogranin Bpeptide. In aspects of this embodiment, a binding element comprising achromogranin B peptide is derived from a GAWK peptide, anadrenomedullary peptide or a secretolytin. In other aspects of thisembodiment, a binding element comprising a chromogranin B peptidecomprises SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104or SEQ ID NO: 105.

In other aspects of this embodiment, a binding element comprising achromogranin B peptide has, e.g., at least 70% amino acid identity withSEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ IDNO: 105, at least 75% amino acid identity with SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105, at least 80%amino acid identity with SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103,SEQ ID NO: 104 or SEQ ID NO: 105, at least 85% amino acid identity withSEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ IDNO: 105, at least 90% amino acid identity with SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105 or at least95% amino acid identity with SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104 or SEQ ID NO: 105. In yet other aspects of thisembodiment, a binding element comprising a chromogranin B peptide has,e.g., at most 70% amino acid identity with SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105, at most 75% aminoacid identity with SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104 or SEQ ID NO: 105, at most 80% amino acid identity with SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105, at most 85% amino acid identity with SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105, at most 90% aminoacid identity with SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104 or SEQ ID NO: 105 or at most 95% amino acid identity with SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105.

In other aspects of this embodiment, a binding element comprising achromogranin B peptide has, e.g., at least one, two, three, four, five,six, seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:104 or SEQ ID NO: 105. In other aspects of this embodiment, a bindingelement comprising a chromogranin B peptide has, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid substitutions relative to SEQ ID NO: 101, SEQ ID NO: 102, SEQ IDNO: 103, SEQ ID NO: 104 or SEQ ID NO: 105. In yet other aspects of thisembodiment, a binding element comprising a chromogranin B peptide has,e.g., at least one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid deletions relative to SEQ ID NO: 101, SEQID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105. In yetother aspects of this embodiment, a binding element comprising achromogranin B peptide has, e.g., at most one, two, three, four, five,six, seven, eight, nine or ten non-contiguous amino acid deletionsrelative to SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:104 or SEQ ID NO: 105. In still other aspects of this embodiment, abinding element comprising a chromogranin B peptide has, e.g., at leastone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105. In yet otheraspects of this embodiment, a binding element comprising a chromograninB peptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid additions relative to SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105.

In other aspects of this embodiment, a binding element comprising achromogranin B peptide has, e.g., at least one, two, three, four, five,six, seven, eight, nine or ten contiguous amino acid substitutionsrelative to SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:104 or SEQ ID NO: 105. In other aspects of this embodiment, a bindingelement comprising a chromogranin B peptide has, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104 or SEQ ID NO: 105. In yet other aspects of thisembodiment, a binding element comprising a chromogranin B peptide has,e.g., at least one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid deletions relative to SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105. In yet otheraspects of this embodiment, a binding element comprising a chromograninB peptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten contiguous amino acid deletions relative to SEQ IDNO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105. In still other aspects of this embodiment, a binding elementcomprising a chromogranin B peptide has, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103,SEQ ID NO: 104 or SEQ ID NO: 105. In yet other aspects of thisembodiment, a binding element comprising a chromogranin B peptide has,e.g., at most one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid additions relative to SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105.

In another embodiment, a binding element comprising a chromogranin Cpeptide. In aspects of this embodiment, a binding element comprising achromogranin C peptide is derived from a secretoneurin. In other aspectsof this embodiment, a binding element comprising a chromogranin Cpeptide comprises SEQ ID NO: 106.

In other aspects of this embodiment, a binding element comprising achromogranin C peptide has, e.g., at least 70% amino acid identity withSEQ ID NO: 106, at least 75% amino acid identity with SEQ ID NO: 106, atleast 80% amino acid identity with SEQ ID NO: 106, at least 85% aminoacid identity with SEQ ID NO: 106, at least 90% amino acid identity withSEQ ID NO: 106 or at least 95% amino acid identity with SEQ ID NO: 106.In yet other aspects of this embodiment, a binding element comprising achromogranin C peptide has, e.g., at most 70% amino acid identity withSEQ ID NO: 106, at most 75% amino acid identity with SEQ ID NO: 106, atmost 80% amino acid identity with SEQ ID NO: 106, at most 85% amino acididentity with SEQ ID NO: 106, at most 90% amino acid identity with SEQID NO: 106 or at most 95% amino acid identity with SEQ ID NO: 106.

In other aspects of this embodiment, a binding element comprising achromogranin C peptide has, e.g., at least one, two, three, four, five,six, seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to SEQ ID NO: 106. In other aspects of this embodiment, abinding element comprising a chromogranin C peptide has, e.g., at mostone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid substitutions relative to SEQ ID NO: 106. Inyet other aspects of this embodiment, a binding element comprising achromogranin C peptide has, e.g., at least one, two, three, four, five,six, seven, eight, nine or ten non-contiguous amino acid deletionsrelative to SEQ ID NO: 106. In yet other aspects of this embodiment, abinding element comprising a chromogranin C peptide has, e.g., at mostone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid deletions relative to SEQ ID NO: 106. In stillother aspects of this embodiment, a binding element comprising achromogranin C peptide has, e.g., at least one, two, three, four, five,six, seven, eight, nine or ten non-contiguous amino acid additionsrelative to SEQ ID NO: 106. In yet other aspects of this embodiment, abinding element comprising a chromogranin C peptide has, e.g., at mostone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 106.

In other aspects of this embodiment, a binding element comprising achromogranin C peptide has, e.g., at least one, two, three, four, five,six, seven, eight, nine or ten contiguous amino acid substitutionsrelative to SEQ ID NO: 106. In other aspects of this embodiment, abinding element comprising a chromogranin C peptide has, e.g., at mostone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid substitutions relative to SEQ ID NO: 106. In yet otheraspects of this embodiment, a binding element comprising a chromograninC peptide has, e.g., at least one, two, three, four, five, six, seven,eight, nine or ten contiguous amino acid deletions relative to SEQ IDNO: 106. In yet other aspects of this embodiment, a binding elementcomprising a chromogranin C peptide has, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 106. In still other aspects of thisembodiment, a binding element comprising a chromogranin C peptide has,e.g., at least one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid additions relative to SEQ ID NO: 106. In yetother aspects of this embodiment, a binding element comprising achromogranin C peptide has, e.g., at most one, two, three, four, five,six, seven, eight, nine or ten contiguous amino acid additions relativeto SEQ ID NO: 106.

Another example of a binding element disclosed in the presentspecification is, e.g., a tachykinin peptide, such as, e.g., a SubstanceP, a neuropeptide K (NPK), a neuropeptide gamma (NP gamma), a neurokininA (NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B(NKB), a hemokinin or a endokinin. Thus, in an embodiment, a bindingelement is derived from a tachykinin peptide.

In aspects of this embodiment, a binding element comprising a tachykininpeptide is derived from a Substance P, a neuropeptide K (NPK), aneuropeptide gamma (NP gamma), a neurokinin A (NKA; Substance K,neurokinin alpha, neuromedin L), a neurokinin B (NKB), a hemokinin or aendokinin. In other aspects of this embodiment, a binding elementcomprising a tachykinin peptide comprises SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 or SEQ ID NO: 118.

In other aspects of this embodiment, a binding element comprising atachykinin peptide has, e.g., at least 70% amino acid identity with SEQID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118, at least 75% amino acididentity with SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118, at least80% amino acid identity with SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO:118, at least 85% amino acid identity with SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 OR SEQ ID NO: 118, at least 90% amino acid identity with SEQ ID NO:107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:116, SEQ ID NO: 117 OR SEQ ID NO: 118 or at least 95% amino acididentity with SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118. In yetother aspects of this embodiment, a binding element comprising atachykinin peptide has, e.g., at most 70% amino acid identity with SEQID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118, at most 75% amino acididentity with SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118, at most80% amino acid identity with SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO:118, at most 85% amino acid identity with SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 OR SEQ ID NO: 118, at most 90% amino acid identity with SEQ ID NO:107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:116, SEQ ID NO: 117 OR SEQ ID NO: 118 or at most 95% amino acid identitywith SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO:115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118.

In other aspects of this embodiment, a binding element comprising atachykinin peptide has, e.g., at least one, two, three, four or fivenon-contiguous amino acid substitutions relative to SEQ ID NO: 107, SEQID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQID NO: 117 OR SEQ ID NO: 118. In other aspects of this embodiment, abinding element comprising a tachykinin peptide has, e.g., at most one,two, three, four or five non-contiguous amino acid substitutionsrelative to SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118. In yetother aspects of this embodiment, a binding element comprising atachykinin peptide has, e.g., at least one, two, three, four or fivenon-contiguous amino acid deletions relative to SEQ ID NO: 107, SEQ IDNO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112,SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ IDNO: 117 OR SEQ ID NO: 118. In yet other aspects of this embodiment, abinding element comprising a tachykinin peptide has, e.g., at most one,two, three, four or five non-contiguous amino acid deletions relative toSEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ IDNO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115,SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118. In still other aspectsof this embodiment, a binding element comprising a tachykinin peptidehas, e.g., at least one, two, three, four or five non-contiguous aminoacid additions relative to SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO:118. In yet other aspects of this embodiment, a binding elementcomprising a tachykinin peptide has, e.g., at most one, two, three, fouror five non-contiguous amino acid additions relative to SEQ ID NO: 107,SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ IDNO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116,SEQ ID NO: 117 OR SEQ ID NO: 118.

In other aspects of this embodiment, a binding element comprising atachykinin peptide has, e.g., at least one, two, three, four or fivecontiguous amino acid substitutions relative to SEQ ID NO: 107, SEQ IDNO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112,SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ IDNO: 117 OR SEQ ID NO: 118. In other aspects of this embodiment, abinding element comprising a tachykinin peptide has, e.g., at most one,two, three, four or five contiguous amino acid substitutions relative toSEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ IDNO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115,SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118. In yet other aspectsof this embodiment, a binding element comprising a tachykinin peptidehas, e.g., at least one, two, three, four or five contiguous amino aciddeletions relative to SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109,SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ IDNO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO:118. In yet other aspects of this embodiment, a binding elementcomprising a tachykinin peptide has, e.g., at most one, two, three, fouror five contiguous amino acid deletions relative to SEQ ID NO: 107, SEQID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQID NO: 117 OR SEQ ID NO: 118. In still other aspects of this embodiment,a binding element comprising a tachykinin peptide has, e.g., at leastone, two, three, four or five contiguous amino acid additions relativeto SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO:115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ ID NO: 118. In yet otheraspects of this embodiment, a binding element comprising a tachykininpeptide has, e.g., at most one, two, three, four or five contiguousamino acid additions relative to SEQ ID NO: 107, SEQ ID NO: 108, SEQ IDNO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113,SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 OR SEQ IDNO: 118.

Another example of a binding element disclosed in the presentspecification is, e.g., a Neuropeptide Y related peptide, such as, e.g.,a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide (PP) or aPancreatic icosapeptide (PIP). Thus, in an embodiment, a binding elementis derived from a Neuropeptide Y related peptide.

In aspects of this embodiment, a binding element comprising aNeuropeptide Y related peptide is derived from a Neuropeptide Y (NPY), aPeptide YY (PYY), Pancreatic peptide (PP) or a Pancreatic icosapeptide(PIP). In other aspects of this embodiment, a binding element comprisinga Neuropeptide Y related peptide comprises SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123.

In other aspects of this embodiment, a binding element comprising aNeuropeptide Y related peptide has, e.g., at least 70% amino acididentity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122 or SEQ ID NO: 123, at least 75% amino acid identity with SEQ ID NO:119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123,at least 80% amino acid identity with SEQ ID NO: 119, SEQ ID NO: 120,SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123, at least 85% aminoacid identity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQID NO: 122 or SEQ ID NO: 123, at least 90% amino acid identity with SEQID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123 or at least 95% amino acid identity with SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123. In yet otheraspects of this embodiment, a binding element comprising a NeuropeptideY related peptide has, e.g., at most 70% amino acid identity with SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123, at most 75% amino acid identity with SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123, at most 80% aminoacid identity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQID NO: 122 or SEQ ID NO: 123, at most 85% amino acid identity with SEQID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123, at most 90% amino acid identity with SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123 or at most 95%amino acid identity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121,SEQ ID NO: 122 or SEQ ID NO: 123.

In other aspects of this embodiment, a binding element comprising aNeuropeptide Y related peptide has, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122 or SEQ ID NO: 123. In other aspects of thisembodiment, a binding element comprising a Neuropeptide Y relatedpeptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid substitutions relative toSEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ IDNO: 123. In yet other aspects of this embodiment, a binding elementcomprising a Neuropeptide Y related peptide has, e.g., at least one,two, three, four, five, six, seven, eight, nine or ten non-contiguousamino acid deletions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ IDNO: 121, SEQ ID NO: 122 or SEQ ID NO: 123. In yet other aspects of thisembodiment, a binding element comprising a Neuropeptide Y relatedpeptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid deletions relative to SEQID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123. In still other aspects of this embodiment, a binding elementcomprising a Neuropeptide Y related peptide has, e.g., at least one,two, three, four, five, six, seven, eight, nine or ten non-contiguousamino acid additions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ IDNO: 121, SEQ ID NO: 122 or SEQ ID NO: 123. In yet other aspects of thisembodiment, a binding element comprising a Neuropeptide Y relatedpeptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten non-contiguous amino acid additions relative to SEQID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123.

In other aspects of this embodiment, a binding element comprising aNeuropeptide Y related peptide has, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122 or SEQ ID NO: 123. In other aspects of thisembodiment, a binding element comprising a Neuropeptide Y relatedpeptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten contiguous amino acid substitutions relative to SEQID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123. In yet other aspects of this embodiment, a binding elementcomprising a Neuropeptide Y related peptide has, e.g., at least one,two, three, four, five, six, seven, eight, nine or ten contiguous aminoacid deletions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122 or SEQ ID NO: 123. In yet other aspects of thisembodiment, a binding element comprising a Neuropeptide Y relatedpeptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten contiguous amino acid deletions relative to SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123. In still other aspects of this embodiment, a binding elementcomprising a Neuropeptide Y related peptide has, e.g., at least one,two, three, four, five, six, seven, eight, nine or ten contiguous aminoacid additions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122 or SEQ ID NO: 123. In yet other aspects of thisembodiment, a binding element comprising a Neuropeptide Y relatedpeptide has, e.g., at most one, two, three, four, five, six, seven,eight, nine or ten contiguous amino acid additions relative to SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123.

It is envisioned that a modified Clostridial toxin disclosed in thepresent specification can comprise a binding element in any and alllocations with the proviso that modified Clostridial toxin is capable ofperforming the intoxication process. Non-limiting examples include,locating a binding element at the amino terminus of a modifiedClostridial toxin; locating a binding element between a Clostridialtoxin therapeutic element and a translocation element of a modifiedClostridial toxin; and locating a binding element at the carboxylterminus of a modified Clostridial toxin. Other non-limiting examplesinclude, locating a binding element between a Clostridial toxinenzymatic domain and a Clostridial toxin translocation domain of amodified Clostridial toxin. The enzymatic domain of naturally-occurringClostridial toxins contains the native start methionine. Thus, in domainorganizations where the enzymatic domain is not in the amino-terminallocation an amino acid sequence comprising the start methionine shouldbe placed in front of the amino-terminal domain. Likewise, where abinding element is in the amino-terminal position, an amino acidsequence comprising a start methionine and a protease cleavage site maybe operably-linked in situations in which a binding element requires afree amino terminus, see, e.g., Shengwen Li et al., DegradableClostridial Toxins, U.S. patent application Ser. No. 11/572,512 (Jan.23, 2007), which is hereby incorporated by reference in its entirety. Inaddition, it is known in the art that when adding a polypeptide that isoperably-linked to the amino terminus of another polypeptide comprisingthe start methionine that the original methionine residue can bedeleted.

Thus, in an embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising a bindingelement, a translocation element, an exogenous protease cleavage siteand a therapeutic element (FIG. 20A). In an aspect of this embodiment, amodified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising a binding element, a Clostridialtoxin translocation domain, an exogenous protease cleavage site and aClostridial toxin enzymatic domain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising a bindingelement, a therapeutic element, an exogenous protease cleavage site, anda translocation element (FIG. 20B). In an aspect of this embodiment, amodified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising a binding element, a Clostridialtoxin enzymatic domain, an exogenous protease cleavage site, aClostridial toxin translocation domain.

In yet another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising atherapeutic element, an exogenous protease cleavage site, a bindingelement, and a translocation element (FIG. 21A). In an aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxinenzymatic domain, an exogenous protease cleavage site, a bindingelement, and a Clostridial toxin translocation domain.

In yet another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising atranslocation element, an exogenous protease cleavage site, a bindingelement, and a therapeutic element (FIG. 21B). In an aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxintranslocation domain, a binding element, an exogenous protease cleavagesite and a Clostridial toxin enzymatic domain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising atherapeutic element, a binding element, an exogenous protease cleavagesite, and a translocation element (FIG. 21C). In an aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxinenzymatic domain, a binding element, an exogenous protease cleavagesite, a Clostridial toxin translocation domain.

In yet another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising atranslocation element, a binding element, an exogenous protease cleavagesite and a therapeutic element (FIG. 21D). In an aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxintranslocation domain, a binding element, an exogenous protease cleavagesite and a Clostridial toxin enzymatic domain.

In still another embodiment, a modified Clostridial toxin can comprisean amino to carboxyl single polypeptide linear order comprising atherapeutic element, an exogenous protease cleavage site, atranslocation element, and a binding element (FIG. 22A). In an aspect ofthis embodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxinenzymatic domain, an exogenous protease cleavage site, a Clostridialtoxin translocation domain, and a binding element.

In still another embodiment, a modified Clostridial toxin can comprisean amino to carboxyl single polypeptide linear order comprising atranslocation element, an exogenous protease cleavage site, atherapeutic element and a binding element, (FIG. 22B). In an aspect ofthis embodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxintranslocation domain, a binding element, an exogenous protease cleavagesite and a Clostridial toxin enzymatic domain.

In a particularly preferred embodiment, the single-chain neurotoxin orneurotoxin derivative of the invention, altered as indicated above, isfurther modified to remove other incidental endogenous proteolytic sitessuch as those cleaved by trypsin, Arg C protease, chymotrypsin, or hostcell proteases. As indicated below, modification of the primary aminoacid sequences in these regions to confer protease resistance canincrease the yield of the neurotoxin and reduce the toxicity of thesingle-chain neurotoxin prior to cleavage and activation.

In another preferred embodiment, the recombinant modified single-chainneurotoxin is further modified by joining the chain to a binding tagcomprising one member of a specific binding complex. By “specificbinding complex” is meant two or more chemical or biochemical entitiesthat will bind each other under defined environmental conditions andwhich will not significantly bind other chemical or biochemical entitiespresent in the environment under the same conditions. Examples ofmembers of a specific binding complex include, without limitation, anantibody and its antigen, a lectin and its target carbohydrate, anucleic acid strand and its complementary nucleic acid strand, a cellsurface receptor and its ligand, a metal and a compound able to form acoordination or chelation complex with that metal, and the like.

In this embodiment, the binding tag may be joined to the single-chaintoxin through a linker, preferably a cleavable linker. Examples ofpossible linkers, while not an exhaustive list, include 1) aliphaticdicarboxylic acids of the formula HOOC—(CH₂)_(n)—COOH, where n=1-12 (maybe linked at a free amino group); 2) HO—(CH₂)_(n)—COOH, where n>10(suitable for attachment at the amino terminus of the polypeptide), 3)substituted polybenzene structures, and 4) a N-hydroxysuccinimide (NHS)ester linker. The use of an linker containing an ester permits cleavageof the ester linker following use in the purification of thesingle-chain neurotoxin under relatively mild acidic conditions.

Alternatively, and most preferably, the binding tag may comprise some orall of the amino acid sequence of an appropriately chosen polypeptidecoexpressed with the single-chain toxin as a fusion protein; suchpolypeptides may comprise, without limitation, the maltose bindingdomain of maltose binding protein (MBP), a polyhistidine peptide likeHIS₆, the calmodilin binding domain of calmodulin binding protein, theglutathione binding domain of glutathione-S-transferase, FLAG, humanInfluenza virus hemagluttinin (HA), human p62c-Myc protein (c MYC),Vesicular Stomatitis Virus Glycoprotein (VSV-G), Substance P,glycoprotein-D precursor of Herpes simplex virus (HSV), V5, AU1 and AU5,streptavidin binding peptide (strep), and biotin or a biotinylationsequence. Non-limiting examples of specific protocols for selecting,making and using an appropriate binding peptide are described in, e.g.,Epitope Tagging, pp. 17.90-17.93 (Sambrook and Russell, eds., MolecularCloning A Laboratory Manual, Vol. 3, 3rd ed. 2001); Antibodies: ALaboratory Manual (Edward Harlow & David Lane, eds., Cold Spring HarborLaboratory Press, 2nd ed. 1998); and Using Antibodies: A LaboratoryManual Portable Protocol No. I (Edward Harlow & David Lane, Cold SpringHarbor Laboratory Press, 1998). In addition, non-limiting examples ofbinding tags as well as well-characterized reagents, conditions andprotocols are readily available from commercial vendors that include,without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BDBiosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif.These protocols are routine procedures well within the scope of oneskilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise a binding tag. In anotherembodiment, a modified Clostridial toxin disclosed in the presentspecification can further comprises a plurality of binding tags. Inaspects of this embodiment, a modified Clostridial toxin can comprise,e.g., at least 1 binding tag, at least 2 binding tags, at least 3binding tags, at least 4 binding tags or at least 5 binding tags. Inother aspects of this embodiment, a modified Clostridial toxin cancomprise, e.g., at most 1 binding tag, at most 2 binding tags, at most 3binding tags, at most 4 binding tags or at most 5 binding tags. Inanother aspect of this embodiment, a modified Clostridial toxin cancomprise one or more copies of the same binding tag, one or more copiesof different binding tag, or any combination thereof.

The location of a binding tag can be in various positions, including,without limitation, at the amino terminus of a modified Clostridialtoxin, within a modified Clostridial toxin, or at the carboxyl terminusof a modified Clostridial toxin. Thus, in an embodiment, a binding tagis located at the amino-terminus of a modified Clostridial toxin. Insuch a location, a start methionine should be placed in front of thebinding tag. In addition, it is known in the art that when adding apolypeptide that is operably-linked to the amino terminus of anotherpolypeptide comprising the start methionine that the original methionineresidue can be deleted. This is due to the fact that the addedpolypeptide will contain a new start methionine and that the originalstart methionine may reduce optimal expression of the fusion protein. Inaspects of this embodiment, a binding tag located at the amino-terminusof a modified Clostridial toxin disclosed in the present specificationcan be, e.g., a FLAG Express™ binding tag, a human Influenza virushemagluttinin (HA) binding tag, a human p62c-Myc protein (c MYC) bindingtag, a Vesicular Stomatitis Virus Glycoprotein (VSV-G) binding tag, aSubstance P binding tag, a glycoprotein-D precursor of Herpes simplexvirus (HSV) binding tag, a V5 binding tag, a AU1 binding tag, a AU5binding tag, a polyhistidine binding tag, a streptavidin binding peptidebinding tag, a biotin binding tag, a biotinylation binding tag, aglutathione binding domain of glutathione-S-transferase, a calmodulinbinding domain of the calmodulin binding protein or a maltose bindingdomain of the maltose binding protein.

In another embodiment, an epitope-binding region is located at thecarboxyl-terminus of a modified Clostridial toxin. In aspects of thisembodiment, an epitope-binding region located at the carboxyl-terminusof a modified Clostridial toxin disclosed in the present specificationcan be, e.g., a FLAG Express™ binding tag, a human Influenza virushemagluttinin (HA) binding tag, a human p62c-Myc protein (c MYC) bindingtag, a Vesicular Stomatitis Virus Glycoprotein (VSV-G) binding tag, aSubstance P binding tag, a glycoprotein-D precursor of Herpes simplexvirus (HSV) binding tag, a V5 binding tag, a AU1 binding tag, a AU5binding tag, a polyhistidine binding tag, a streptavidin binding peptidebinding tag, a biotin binding tag, a biotinylation binding tag, aglutathione binding domain of glutathione-S-transferase, a calmodulinbinding domain of the calmodulin binding protein or a maltose bindingdomain of the maltose binding protein.

Additionally, the binding tag may be constructed to have a proteasecleavage site between itself and either the amino terminus or thecarboxyl terminus of the single-chain toxin so as be removable followingpurification of the peptide. The proteolytic cleavage site may bedesigned to be cleaved by the same activator protease chosen to nick thesingle-chain toxin between the H and L chains.

It is therefore an object of the invention to provide a recombinantactivatible single-chain neurotoxin molecule that has reduced toxicitycompared to the native neurotoxin until activated by reaction with anon-clostridial protease. The single-chain neurotoxin is more easilypurified, is less dangerous to handle in the purification process, andcan be optionally modified to give the toxin more desirable properties.

It is also an object of the invention to provide an method of making arecombinant activatable single-chain neurotoxin by modifying thenucleotide sequence encoding the neurotoxin to replace the native aminoacid proteolytic cleavage sequence separating the H and L chain with anamino acid sequence stable to indigenous clostridial or host cellproteases but susceptible to cleavage by chosen protease in vitro.

It is further an object of the present invention to provide more stableneurotoxin polypeptides through modification of the nucleotide sequenceof the coding region of the H and L chains thereof, removing incidentalproteolytic cleavage sites by causing the replacement of labile aminoacids with other amino acid residues which confer upon the toxinresistance to undesired proteolytic degradation.

Additionally, it is an object of the invention to provide methods ofpurifying recombinant neurotoxins as a single-chain by joining theexpressed single-chain neurotoxin to a binding moiety comprising partnerof a specific binding complex which can be used in the affinitypurification with the binding partner comprising the other half of thebinding complex. Purification can be performed batch-wise or in achromatography column. The binding moiety may then be removed followingthe affinity step, and separated from the neurotoxin.

It is also an object of the invention to provide single-chainrecombinant modified neurotoxin molecules for use as therapeutic agents.The modified neurotoxin molecules may have an altered target specificityor an altered activity compared to the native neurotoxin from which itis derived, or both.

Another aspect of the present invention provides a method of activatingan activatable polypeptide disclosed in the present specification, suchmethod comprising the step of incubating the activatable polypeptidewith an exogenous protease, wherein the exogenous protease can cleavethe exogenous protease cleavage site present in the polypeptide andwherein cleavage of the activatable polypeptide by the exogenousprotease converts the activatable polypeptide from its single-chainpolypeptide form into its di-chain form, thereby activating thepolypeptide.

Aspects of the present invention provide, in part, an exogenousprotease. As used herein, the term “exogenous protease” means anyprotease capable of selectively cleaving the P1-P1′ scissile bondcomprising the exogenous protease cleavage site, with the proviso thatthe exogenous protease is not a human protease or a protease beingexpressed by the host cell expressing a construct encoding anactivatable polypeptide disclosed in the present specification. As usedherein, the term “selectively” means having a highly preferred activityor effect. Thus, with reference to an exogenous protease, there is adiscriminatory proteolytic cleavage of the P1-P1′ scissile bondcomprising the exogenous protease cleavage site. It is envisioned thatany and all proteases capable of selectively cleaving the P1-P1′scissile bond comprising the exogenous protease cleavage site can beuseful in the disclosed methods. As a non-limiting example, a bovineenterokinse can selectively cleave a bovine enterokinse cleavage site, atobacco etch virus protease can selectively cleave a tobacco etch virusprotease cleavage site, a human rhinovirus 3C protease can selectivelycleave a human rhinovirus 3C protease cleavage site, a subtilisin canselectively cleave a subtilisin cleavage site, a hydroxylamine canselectively cleave a hydroxylamine cleavage site, and a SUMO/ULP-1protease can selectively cleave a SUMO/ULP-1 protease cleavage site.

A therapeutic agent useful in the invention generally is administered asa pharmaceutical acceptable composition comprising a modified neurotoxinas disclosed in the present specification. As used herein, the term“pharmaceutically acceptable” means any molecular entity or compositionthat does not produce an adverse, allergic or other untoward or unwantedreaction when administered to an individual. As used herein, the term“pharmaceutically acceptable composition” is synonymous with“pharmaceutical composition” and means a therapeutically effectiveconcentration of an active ingredient, such as, e.g., any of themodified neurotoxins disclosed in the present specification. Apharmaceutical composition comprising a modified neurotoxin is usefulfor medical and veterinary applications. A pharmaceutical compositionmay be administered to a patient alone, or in combination with othersupplementary active ingredients, agents, drugs or hormones. Thepharmaceutical compositions may be manufactured using any of a varietyof processes, including, without limitation, conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, and lyophilizing. The pharmaceuticalcomposition can take any of a variety of forms including, withoutlimitation, a sterile solution, suspension, emulsion, lyophilizate,tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosageform suitable for administration.

It is also envisioned that a pharmaceutical composition comprising amodified neurotoxin can optionally include a pharmaceutically acceptablecarriers that facilitate processing of an active ingredient intopharmaceutically acceptable compositions. As used herein, the term“pharmacologically acceptable carrier” is synonymous with“pharmacological carrier” and means any carrier that has substantiallyno long term or permanent detrimental effect when administered andencompasses terms such as “pharmacologically acceptable vehicle,stabilizer, diluent, additive, auxiliary or excipient.” Such a carriergenerally is mixed with an active compound, or permitted to dilute orenclose the active compound and can be a solid, semi-solid, or liquidagent. It is understood that the active ingredients can be soluble orcan be delivered as a suspension in the desired carrier or diluent. Anyof a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., water,saline, glycine, hyaluronic acid and the like; solid carriers such as,e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like;solvents; dispersion media; coatings; antibacterial and antifungalagents; isotonic and absorption delaying agents; or any other inactiveingredient. Selection of a pharmacologically acceptable carrier candepend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activeingredient, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS (Howard C. Ansel et al., eds., Lippincott Williams& Wilkins Publishers, 7 ed. 1999); REMINGTON: THE SCIENCE AND PRACTICEOF PHARMACY (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, 20ed. 2000); GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS(Joel G. Hardman et al., eds., McGraw-Hill Professional, 10th ed. 2001);and HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (Raymond C. Rowe et al., APhAPublications, 4^(th) edition 2003). These protocols are routineprocedures and any modifications are well within the scope of oneskilled in the art and from the teaching herein.

It is further envisioned that a pharmaceutical composition disclosed inthe present specification can optionally include, without limitation,other pharmaceutically acceptable components (or pharmaceuticalcomponents), including, without limitation, buffers, preservatives,tonicity adjusters, salts, antioxidants, osmolality adjusting agents,physiological substances, pharmacological substances, bulking agents,emulsifying agents, wetting agents, sweetening or flavoring agents, andthe like. Various buffers and means for adjusting pH can be used toprepare a pharmaceutical composition disclosed in the presentspecification, provided that the resulting preparation ispharmaceutically acceptable. Such buffers include, without limitation,acetate buffers, citrate buffers, phosphate buffers, neutral bufferedsaline, phosphate buffered saline and borate buffers. It is understoodthat acids or bases can be used to adjust the pH of a composition asneeded. Pharmaceutically acceptable antioxidants include, withoutlimitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Usefulpreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate, a stabilized oxy chloro composition, such as, e.g., PURITE® andchelants, such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, andCaNaDTPA-bisamide. Tonicity adjustors useful in a pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor. The pharmaceuticalcomposition may be provided as a salt and can be formed with many acids,including but not limited to, hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents than are the corresponding free base forms.It is understood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical composition useful inthe invention.

In an embodiment, a therapeutic agent is a pharmaceutical compositioncomprising a modified neurotoxin. In an aspect of this embodiment, apharmaceutical composition comprises an unactivated, single-chain for ofthe modified toxin. In another aspect of this embodiment, apharmaceutical composition comprises an activated di-chain form of themodified toxin. In other aspects of this embodiment, a pharmaceuticalcomposition comprising a modified neurotoxin further comprises apharmacological carrier, a pharmaceutical component, or both apharmacological carrier and a pharmaceutical component. In other aspectsof this embodiment, a pharmaceutical composition comprising a modifiedneurotoxin further comprises at least one pharmacological carrier, atleast one pharmaceutical component, or at least one pharmacologicalcarrier and at least one pharmaceutical component.

It is also an object of the invention to provide a single-chainactivatable recombinant neurotoxin that may be more easily purified thanthe wild type neurotoxin. Such a neurotoxin permits the large scalepreparation of properly folded highly pure toxin for clinical use.

EXAMPLES

The following Examples serve to illustrate particular embodiments of theinvention, and do not limit the scope of the invention defined in theclaims in any way.

Example 1 Construction of an Expression Vector Containing a Single-ChainTeNT Coding Region

The present invention can be exemplified describing the construction ofa plasmid that will express TeNT in E. coli as a single protein that isreadily purified, i.e., by affinity chromatography. TeNT can be chosenas a pilot system because (i) the availability of an excellent vaccinegreatly reduces the risk of its handling and (ii) it is the mostcomprehensively studied of the toxins in terms of expressing HC and LCdomains. However, those of skill in the art will understand that thesame or similar strategies may be employed using any di-chain or binarytoxin or other bioactive molecule expressed as a single polypeptide andactivated by proteolytic cleavage. Single chain molecules wereconstructed containing the wild type TeNT L chain and a mutated versionof the TeNT light chain wherein a glutamic acid residue at position 234is changed to an alanine (termed “E234A”, Ala²³⁴, or “the E234A mutantlight chain”), respectively. This latter mutation results in an inactiveTeNT light chain, and a plasmid encoding the E234A mutant light chain(pMAL-E234A) was constructed as described in Li et al., Biochemistry33:7014-7020 (1994) (hereby incorporated by reference herein). Thefollowing protocol is used for the construction of each single-chaintoxin.

The vector pTrcHisA, purchased from Invitrogen, is modified using aStratagene QuickChange® site-directed mutagenesis kit (for site-directedmutagenesis techniques, see e.g., Smith et al., J. Biol. Chem.253:6651-6560 (1979); incorporated by reference herein in its entirety)to create two extra restriction sites (SalI and HindIII) upstream of thenucleotides encoding a pre-existing enterokinase (EK) cleavage site. Theplasmid also contains a translational start codon (ATG) followed by arun of codons encoding 6 histidine residues immediately upstream of theenterokinase cleavage site. A multiple cloning site containing Bam HI,XhoI, Bgl II, Pst I, Kpn I, Eco RI BstB I and Hind III cleavage sites islocated immediately downstream of the EK site; the Hind III site isremoved by site-directed mutagenesis. The following primers are employedto insert the restriction sites (underlined) upstream of the EK cleavagesite: (SEQ ID NO: 67) GACTGGTGGACAGCAAGTCGACCGGAAGCTTTACGACGATGACG,                Sal I  Hind III and (SEQ ID NO: 68)CGTCATCGTCGTAAAGCTTCCGGTCGACTTGCTGTCCACCAGTC             Hind III  Sal I

The resulting plasmid contains both Sal I and Hind III sites located atthe 5′ side of the nucleotide sequence encoding the bovine enterokinase(EK) cleavage site.

The nucleotide sequence encoding the wild-type TeNT L chain is obtainedfrom plasmid pMAL-LC, described in Li et al., Biochemistry 33, 7014-7020(1994), incorporated by reference herein. The plasmid encodes the TeNTlight chain as a fusion protein with maltose binding protein (MBP)located immediately upstream of the coding sequence for the L chain. TheMBP and L chain portions of the fusion protein are designed to containthe cleavage site for human blood coagulation factor Xa(Ile-Glu-Gly-Arg) to facilitate removal of the MBP once affinitypurification has been performed.

The DNA fragment containing the coding sequence of the L chain isexcised from plasmid pMAL-LC by digesting the plasmid with Sal I andHind III, gel purifying the resulting DNA fragment containing the Lchain, and ligating this fragment into plasmid pTrcHisA at the newlycreated Sal I and Hind III sites upstream of the EK site. This fragmentresults in the excission of the maltose binding protein sequences fromthe N terminus of the L chain.

An identical procedure is used to subclone the DNA fragment containing amutant L chain from plasmid pMAL-LC-Ala²³⁴, in which a single amino acidchange is made at amino acid 234 of the L chain, substituting the nativeglutamic acid with alanine. This change is sufficient to abrogate thezinc endopeptidase activity of the L chain, and to render non-toxic areconstituted tetanus toxin containing native H chain and the Ala²³⁴ Lchain.

The DNA fragment containing the H chain is obtained from plasmidpMAL-HC; construction of this vector is described in Li et al., J.Biochem. 125:1200-1208 (1999), hereby incorporated by reference herein.Briefly, the gene encoding the H chain is constructed by assemblingthree DNA fragments containing different portions of the H chain codingsequence which had been cloned into separate plasmids. The fragmentscomprising the amino terminal half of the H are first amplified usingstandard polymerase chain reaction methods (see, e.g., Mullis, U.S. Pat.No. 4,683,202 and Mullis et al., U.S. Pat. No. 4,800,159, bothincorporated by reference herein in their entirety) and the followingprimers: PCR primers a (containing a Xba I cleavage site) and b(containing a Bgl II cleavage site) (SEQ ID NO: 113 and 114,respectively) are used to amplify the H chain fragment contained in aplasmid termed pTet8; PCR primers c (containing a Bgl II cleavage site)and d (containing both a Hind III and a Sal I cleavage site) (SEQ ID NO:115 and 116, respectively) are used to amplify the H chain fragmentcontained in a plasmid termed pTet14. The nucleotide sequences of theseprimer are provided below, with restriction sites underlined.AATAGATCTAGATCATTAACAGATTTAGGA (a) (SEQ ID NO: 69)TTCTAAAGATCTATACATTTGATAACT (b) (SEQ ID NO: 70)ATGTATAGATCTTTAGAATATCAAGTA (c) (SEQ ID NO: 71)ATCGATAAGCTTTTATCAGTCGACCCAACA (d) (SEQ ID NO: 72) ATCCAGATTTTTAGA

Following PCR amplification and gel purification of the amplified Hchain fragments, each fragment is digested with Bgl II and ligated toyield the complete N terminal half of the H chain coding region. Thisligation product is then digested with Xba I and Hind III and subclonedinto the multiple cloning site of pMAL-c2-T (the plasmid being also cutwith Xba I and Hind III), which is located downstream of the codingregion for MBP and the factor Xa site. pMAL-c2 is a commerciallyavailable vector well known to those of skill in the art. The resultingplasmid is pMAL-H_(N).

The entire H chain coding region is assembled as follows. The pMAL-H_(N)plasmid is digested with Sac I and Sal I to yield the DNA fragmentencoding the N-terminus of the H chain. Plasmid pTet215 is digested withSal I and Bam HI to yield the DNA fragment encoding the H chain carboxylterminus. The vector pMAL-c2-T is digested with Sac I and Bam HI, andligated to the digested H chain fragments, which will assemble in theproper orientation due to the use of distinct endonucleases. Theresulting plasmid is pMAL-HC.

The DNA fragments encoding the H and L chains (including Ala²³⁴ L chain)are cut and purified directly from pMAL-LC or pMALE234A and pMAL-HCconstructs and subcloned into the modified pTrcHisA vector describedabove. The H chain was first ligated into the modified vector at the BamHI site immediately downstream of the EK site, and the resulting plasmidwas gel purified. Following digestion of this plasmid with Hind III andSal I, the L chain was ligated at a position just upstream of the EKcleavage site.

The resulting plasmid construct contains the nucleotide sequenceencoding the single-chain toxin protein, comprising (from amino tocarboxyl terminus): six histidine residues (the His tag), followed bythe L chain, an enterokinase cleavage site, and the H chain. Thetranslated junction between the L and H chains containing the EKcleavage site (SEQ ID NO: 21) is shown below (in the direction fromN-terminus to C-terminus) and in FIG. 1. SEQ ID NO: 73                              EK siteSKLIGLCKKIIPPTNIRENLYNRTA-GEKLYDDDDKDRWGSSR-SLTDLG         Lchain            interchain loop GELCIKNEDLTFIAEKN  H chain

To allow expression of the two chains as a single unit, a nucleotidesequence comprising a stop codon present at the 3′ end of the L chaincoding sequence in the pMAL-LC is removed by site-directed mutagenesisusing two primers (SEQ ID NO: 74 and 75), resulting in a single readingframe containing both H and L chains. (SEQ ID NO: 74)AATAGAACTGCAGGAGAAAAGCTTTACGACGATGAC, TGATAA (deleted stop codon; codingstrand) and (SEQ ID. NO: 75) GTCATCGTCGTAAAGCTTTTCTCCTGCAGTTCTATT TTATCA(deleted stop codon; non-coding strand)

The resulting pTrcHisA-based construct is transformed into E. colistrain JM109 by heat shock using the method of Hanahan, and transformantcolonies are isolated on Luria agar plates containing 100 μg/mlampicillin. Plasmids are purified from these transformants and theinsertions are confirmed by analytical restriction endonucleasedigestion and agarose gel electrophoresis.

Example 2 Expression and Physical Characterization of Single-Chain TeNT

Expression of the pTrcHisA-based single-chain TeNT construct (undercontrol of a hybrid trp/lac promoter) is induced by addition of 1 mMIPTG (isopropyl thio-galactopyranoside) to a confluent culture of arepresentative transformant clone in 200 ml Luria broth containing 100μg/ml ampicillin and incubating further at 37° C. for 16 hours beforecell harvest by centrifugation.

The cell pellets are resuspended in 30 ml Buffer A (20 mM Na₂PO₄, 500 mMNaCl (pH 7.8)), then lysed by ultrasonication at 4° C., using 10-secondbursts at a medium setting. Insoluble debris is removed bycentrifugation at 9,000×g for 30 min at 4° C., and the supernatantrecovered by centrifugation.

The supernatant containing each single-chain construct is incubated for20 minutes at 22° C. with 2 ml of nickel-ion resin (Invitrogen Corp.)for affinity purification by means of chelation between the histidineresidues at the amino terminus of the single-chain toxin molecule andthe nickel. The resins were then load onto mini columns and washed with200 ml of washing buffer (20 mM Na₂PO₄, 500 mM NaCl (pH 6.0)) to removeany non-specifically bound material, the recombinant single-chainproteins are eluted on 0.5 ml fractions with 8-15 ml of 100 mM imidazolein Buffer A. The concentration of the eluted single-chains was measuredby Bradford's protein assay (Bio-Rad Laboratories); approximately 1milligram of the fusion protein was recovered.

Example 3 SDS-PAGE and Western Blot Analysis of Recombinant Single-ChainTeNT

The single-chain TeNT constructs are grown in Luria broth containingampicillin at 37° C., and aliquots taken both before and after inductionof protein expression with IPTG. Crude cell extracts are prepared forSDS-PAGE by dilution in sample buffer under reducing conditions in thepresence of β-mercaptoethanol (BME). Following SDS-PAGE electrophoresis,the separated proteins are Western-blotted as follows: the proteins areelectrophoretically transferred to a polyvinylidenedifluoride (PVDF)membrane using standard methods (see, e.g., Sambrook et al., MolecularCloning, A Laboratory Manual (2d ed. Cold Spring Harbor Laboratory Press1989), hereby incorporated by reference in its entirety), the membranetreated to reduce background Ig binding, and then probed using ananti-His₆ antibody, followed by detection using an alkalinephosphatase-conjugated secondary antibody and development with a5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate.

As shown in lanes 1 and 2 of FIG. 2A, the Western blot revealed nodetectable TeNT expression before induction of protein synthesis; bycontrast, a single band of approximate molecular weight 150 kDa wasrevealed in the aliquots taken following protein induction (See lanes 3and 4.) In FIG. 2A, lanes 1 and 3 are the WT light chain construct andlanes 2 and 4 contain the E234A mutant construct.

FIG. 2B is a Western blot of IPTG-induced cell extracts from cellstransformed with the E234A construct. Significantly, no discernablelower molecular weight proteolytic cleavage products of the light chainwere observed, providing evidence for the relative stability of thesingle-chain toxin following expression and purification.

FIG. 3 shows the results of a second experiment, in which affinitypurified recombinant single-chain (SC) TeNT is nicked with enterokinaseas follows. Thirty micrograms of purified single-chain toxin areincubated with 1 unit of enterokinase in a solution containing 50 mMTris-HCl (pH 8.0), 1 mM CaCl₂ and 0.1% Tween-20 (v/v). As a control, therecombinant protein is incubated in the same reaction mixture containingno EK. These samples, plus an aliquot of native (non-recombinant) TeNTare subjected to SDS-PAGE in an 8% polyacrylamide gel under eitherreducing (+BME) or non-reducing (−BME) conditions. The resulting gel isused both for a Western blot and subsequent detection using anti-H claimantibodies (FIG. 3B), and direct staining with Coomassie Blue (FIG. 3A).

As indicated by FIG. 3, under non-reducing conditions all three samples(Native TeNT (Lane 1), unnicked recombinant toxin (Lane 2), andenterokinase nicked recombinant toxin (Lane 3)) will migrate as doublets(apparently different conformers that resolve into a single band uponreduction) with essentially indistinguishable apparent molecular weightsof about 150 kDa. The non-reducing gel confirms that 1) high levels ofexpression are obtained, 2) the disulfide bonds linking the light andheavy chains are fully formed, and 3) the recombinant single-chain toxinis not subject to observable proteolytic degradation.

By contrast, under reducing conditions wild-type and nicked recombinanttoxin yield an H chain having a molecular weight of about 100 kDa byboth Western blot and Coomassie staining. Additionally, in the Coomassiestained gel, both of these samples also show a lower molecular weightspecies of about 50 kDa, corresponding to the L chain. The wild-type Lchain will migrate with a lower apparent molecular weight than that ofthe recombinant L chain, which has 22 additional amino acid residues dueto the presence of the His₆ moiety and a modified EK cleavagesite-containing interchain junction region. The unnicked recombinanttoxin (Lane 2) migrates as a single band with an apparent molecularweight of about 150 kDa. Notably, no trace of the unnicked toxin is seenin lane 3, indicating the effectiveness of enterokinase treatment.

Example 4 In Vitro Toxin-induced Paralysis by Recombinant Single-ChainTeNT

The biological activity of the recombinant TeNT is also examined andcompared to wild-type toxin using mouse phrenic nerve hemi-diaphragm,since the native toxin is known to cause neuromuscular paralysis, albeitat higher concentrations than act in the CNS. For this experiment, mouseleft phrenic nerve-hemidiaphragm is dissected from mice (T/O strain,4-week old and ˜20 g in weight) and immediately transferred into aclosed circulatory superfusion system containing 10 ml of Krebs-Ringersolution (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 23.8 mMNaHCO₄, 1.2 mM KH₂PO₄, 11.7 mM glucose (pH 7.4)), bubbled with 95% O₂and 5% CO₂ and supplemented with 0.1% (w/v) bovine serum albumin todiminish non-specific adsorption of the toxins (Li et al., Biochemistry33:7014-7020 (1994)). The hemidiaphragms are kept in a bath containing10 ml Krebs-Ringer buffer at 37° C. for 10 minutes before being exposedto 4 or 10 nM native TeNT (▾ and ∇, respectively) or 10 nM nickedrecombinant TeNT (●) or 10 nM un-nicked recombinant TeNT (∘),respectively. (See FIG. 4).

Muscle twitch is evoked by supra-maximal stimulation of the phrenicnerve with bipolar electrodes and recorded via a force-displacementtransducer (Lectromed, UK) connected to an amplifier and computer system(MacLab, AD Instruments, UK). Parameters of nerve stimulation are 0.2 Hzsquare waves of 0.1 msec duration with 1.5-2.5 V amplitude.Toxin-induced paralysis of neuromuscular transmission is quantified asthe time required for nerve-evoked muscle contraction to decrease to 10%(90% reduction) of the original value.

As shown in FIG. 4, 10 nM recombinant nicked TeNT was found to be aspotent as 10 nM native toxin in blocking nerve-induced muscle twitch,with the preparations yielding a 90% reduction in muscle tension inapproximately 170 minutes. Thus, this novel preparation of TeNTexpressed in E. coli at high level as a single-chain, activatablepolypeptide and purified by a simple affinity chromatography step provedto be fully active by all the criteria examined.

By contrast, 10 nM of the unnicked TeNT preparation requireapproximately twice as long to reduce muscle tension, and wasapproximately as active as 4 nM of the wild-type TeNT. As a control,hemidiaphragms incubated with KR buffer and the trace amount ofenterokinase present in the experimental samples were found to shownegligible decrease in muscle tension over 5 hrs.

Thus, this experiment indicates that the unnicked TeNT is considerablyless toxic that either the wild type or recombinant nicked protein invitro.

Example 5 Further Modification of Single-Chain TeNT to RemoveProteolytic Cleavage Sites Reduces Toxicity of Unnicked RecombinantToxin

While the unnicked recombinant single-chain form of TeNT displaysreduced toxicity as compared to the nicked form, the residual toxinactivity probably arises from activation of the toxin by additionalproteases in vivo. To test this possibility, sites in the single-chaintoxin molecule susceptible to proteolytic cleavage by trypsin and Arg Cprotease are identified by incubation of single-chain TeNT with theseenzymes as follows. Fifty micrograms μg of recombinant single-chain TeNTis incubated with 4 μg of Arg-C at 37° C. for 4 h; 0.1 μg of trypsin at37° C. for 0.5 h; or buffer without protease as a control. Thesereactions are terminated by the addition of SDS-PAGE sample buffercontaining 0.1% SDS followed by boiling for 5 minutes; then the samplesare subjected to SDS-PAGE, followed by a Western electrophoretictransfer to a polyvinylidenedifluoride (PVDF) membrane. The membrane isblotted with IgG specific for the His₆-tag and detected using ahorseradish peroxidase staining system.

As shown in FIG. 5, the Western blot reveals that trypsin and Arg Cprotease yielded a L chain (and thus a H chain) fragment of the samesize. Additionally, the transfer of a duplicate gel was stained forprotein with Ponceau red and the H chain band of approximate molecularweight 100 kDa was excised from each lane and analysed by N-terminalsequencing.

In the recombinant single-chain TeNT, the LC and HC are linked by 17amino acids (GEKLYDDDDKDRWGSSR), followed by the beginning of the Hchain sequence (SLTDLGGEL . . . ) N-terminal amino acid sequencing ofthe larger fragment produced by both trypsin and Arg C protease revealthat first 5 amino acids of the 100 kDa trypsin and Arg C proteasecleavage product protein are SLTDL; thus, these proteases appear tocleave the single-chain toxin between the R—S bond (see FIG. 1) so as toliberate the H chain and the L chain containing the EK linker at its Cterminus, with this variant therefore yielding a di-chain toxinessentially identical to the EK nicked toxin.

The arginine at the carboxy terminus of the EK linker sequence ismutated by site-directed mutagenesis to a glycine (R496G), and theresulting single-chain toxin polypeptide is expressed and purified asabove.

Titration of the 6 micrograms of the R496G mutated single-chain (WT LC)toxin and the SC TeNT lacking such a mutation against 0, 0.01, 0.1, 1,10 μg/ml of trypsin, followed by SDS-PAGE and staining with CoomassieBrilliant Blue, yields the cleavage pattern seen in FIG. 6. As can beseen, both single-chain molecules are susceptible to typsin cleavage;however the R496G mutant yields fewer fragments than the SC toxin notcontaining a mutation in the loop region between the chains. Forexample, while three trypsin peptide bands can clearly be seen near thelight chain band upon trypsin cleavage of the SC WT toxin, only two suchbands are seen in the R496G digests.

The fact that there exist remaining trypsin sites in the R496G mutant SCtoxin probably accounts for the fact that this mutant does not cause thelowering of toxicity as compared to the un-nicked SC toxin; bothpreparations give similar values in the mouse lethality andneuromuscular paralysis assays described above.

A different assay system is used to measure neurotoxin activity towardCNS neurons, the cells naturally affected by TeNT. The cells used arecerebellar neurons; these cells are disassociated from the cerebella of7 day old rats. Neurons are suspended at 1-2×10⁶/mL in medium consistingof 3 parts Basal Eagle Medium and 1 part of a buffer consisting of 40 mMHEPES-NaOH (pH 7.3), 78.4 mM KCl, 37.6 mM D-glucose, 2.8 mM CaCl₂, 1.6mM MgSO₄ and 1.0 mM NaH₂PO₄, as well as 1×N2 supplement, 1.0 mML-glutamine, 60 units/mL penicillin, 60 μg/mL streptomycin and 5% (v/v)dialysed horse serum. One milliliter of this cell suspension is added to22 mm diameter poly-D-lysine coated wells. Cytosineβ-D-arabinofuranoside (Ara-C, 40 μM) is added after 20-24 hours in 5%(v/v) CO₂ culture, and neurons are maintained by weekly replacement ofthe above-noted medium containing 40 μM Ara-C.

For each assay, neurons are cultured for at least 10 days in vitro arewashed four times with O₂-gassed Krebs-Ringer HEPES buffer (KRH, mM: 20HEPES.NaOH pH7.4, 128 NaCl, 5 KCl, 1 NaH₂PO₄, 1.4 CaCl₂, 1.2 mM MgSO₄,10 D-glucose and 0.05 mg/mL BSA), and 0.5 mL of the latter buffercontaining 0.25 μCi/mL [14C]-glutamine (i.e. the glutamate precursor) isadded. All steps are performed at 37° C. After a 45 minute labelingperiod, the medium is removed and the neurons washed four times asbefore. Control and toxin-treated neurons are incubated for 5 minutes at37° C. in KRH buffer containing either 1.4 mM Ca²⁺ or 0.5 mM EGTA (i.e.to assess Ca²⁺-independent release); aliquots are then removed andretained for assessment of [¹⁴C]-glutamate content by scintillationcounting. Immediately after removal of the above basal medium, amodified KRH buffer containing 50 mM KCl (containing a lowered 83 mMNaCl content in order to maintain a normal osmotic potential) and either1.4 Ca²⁺ or 0.5 mM EGTA are added for a 5 minute stimulation period.Finally, neurons were solubilized with 20 mM EGTA.NaOH pH 7.5 containing1% (w/v) SDS, and aliquots subjected to scintillation counting in orderto calculate their remaining radioactive contents. The amounts of¹⁴C-glutamate in basal and stimulated samples are expressed aspercentages relative to the calculated total cell content. Thepercentage [¹⁴C]-glutamate contents in EGTA-containing buffer aresubtracted from the values recorded in Ca²⁺-containing samples in orderto calculate the relevant Ca²⁺-dependent component of release and inturn the latter basal readings are subtracted from values obtained for50 mM KCl samples to yield the K⁺-evoked Ca²⁺-dependent glutamaterelease component.

FIG. 8 demonstrates the ability of the recombinant toxin to inhibitneurotransmitter release. Cerebellar neurons, maintained for 10 days invitro, were washed twice with ice-cold KRH buffer containing 5 mM Mg²⁺and 0.5 mM Ca²⁺, then exposed in this buffer to the specifiedconcentrations of (●) native TeNT, (∘) EK-nicked TeNT R496G, (▾)single-chain unnicked TeNT, or (∇) EK-nicked TeNT E234A for 60 min at 4°C. (see FIG. 8). Native TeNT (0.2 nM) was then added to the wellsspecified and, after an additional 30 min, the neurons were washed threetimes with ice-cold KRH buffer and incubated for 30 min at 37° C.Subsequent assessment of K⁺-evoked Ca²⁺-dependent neurotransmitterrelease was performed as detailed above. The results of this assay areshown in FIG. 8.

When cerebellar neurons are exposed to nicked recombinant TeNT, adose-dependent inhibition of Ca⁺⁺ dependent transmitter release is seenwith a potency similar to the native toxin. Nicked recombinant SC TeNT,both WT and R496G, gave similar values in this assay. Thus, while toxinactivity in the unnicked single-chain molecule is not abrogated throughthe removal of a single trypsin cleavage site, the removal of additionalsuch sites is feasible in regions of the single-chain toxin to achievean activatable single-chain proform of the toxin that exhibits evenlower toxicity unless activated in vitro, when its full activity can beachieved.

Example 7 Protease-Deficient TeNT Mutant Antagonises the Actions of TeNTon Peripheral and Central Neurons

Table 3 shows the tabulated results of the indicated TeNT constructstested in three assays of toxin activity: ability to cleave the HV62peptide (which measures proteolytic activity only); neuromuscularparalysis (which is an indication of the toxin molecules' ability toenter the cell and thence to inhibit neurotransmitter release), andmouse lethality upon intraperitoneal injection of the various toxinconstructs. The first two of these assays was performed as describedabove.

The mouse lethality assay was performed essentially as follows: Samplesof recombinant purified single-chain TeNT, R496G mutant TeNT, and E234Amutant TeNT are each divided into two aliquots and one aliquot treatedwith enterokinase to nick the toxin. All samples are serially dilutedinto 50 mM phosphate buffer (pH 7.0), 150 mM NaCl and 0.25% (w/v) bovineserum albumin (BSA), and the toxin preparations are injected into miceintraperitoneally.

As shown in Table 3, the native and nicked TeNT preparations werecomparably active in the mouse lethality assay, having an LD₅₀ of about1×10⁸/mg. The unnicked recombinant toxin and unnicked R496G mutant wereboth about half as active. Finally, the nicked E234A proteolyticallyinactive toxin was less than 5×10⁷ fold less active. TABLE 3 BiologicalActivity of SC TeNT wild type and mutants (E234A and R496G before andafter nicking with enterokinase Time (min.) for 10 nM Initial rete ofcleavage^(a) of to cause 90% Purified TeNT HV62 (nmol · min⁻¹mg⁻¹) Mouselethality^(b) neuromuscular preparations [Relative rate (%)] (LD50/mg)paralysis Native 20.3 ± 0.91 1 × 10⁸ 145 Un-nicked SC WT  8.0 ± 0.03 0.5× 10⁸   260 Nicked^(c) SC WT 22.7 ± 3.37 1 × 10⁸ 150 Un-nicked SC R496G11.7 ± 0.6  0.5 × 10⁸   250 ± 15 Nicked^(c) SC R496G 52.3 ± 4.9  1 × 10⁸135 ± 10 Un-nicked SC E234A ≦0.01^(d) Not tested Not tested Nicked^(c)SC E234A ≦0.01^(d) <50 No detectable activity^(a)Initial rates of proteolysis were measured using the RP-HPLC-basedmethod detailed in Foran et al. (1994). Incubations with 15 μM of asynthetic peptide corresponding to residues 33 to 94 of human VAMP-2(HV62) were performed at 37° C. in 50 mM HEPES, NaOH pH 7.5 containing 2mM DTT 0.2 mg · ml⁻¹ BSA and 50 μM ZnCl₂, using the appropriateconcentration of each reduced toxin preparation required to proteolyze10-15% of the substrate during a 30 min# period. Data are means (± S.D.; n = 4).^(b)LD₅₀ is the amount of toxin that killed 50% of the injected micewithin 4 days.^(c)Toxin preparations were nicked with EK (1 unit/30 μg) at 22° C. for1 h.^(d)This v° value represents the detection limits of the RP-HPLC assay;no proteolysis of HV62 was observed using prolonged incubations.

Purified SC E234A TeNT, in which the catalytic E at position 234 wasreplaced by an A, failed to show any detectable proteolysis of a peptidecontaining residues 33 to 94 of human VAMP-2 (termed HV62), eitherbefore or after nicking with EK. Accordingly, nicked TeNT E234A provedto be devoid of toxicity in mice and unable to inhibit transmitterrelease at the neuromuscular junction or from cerebellar neurons.

Importantly, however, this mutant toxin retained the ability to bind tothe cell surface receptors on peripheral and central neurons.Pre-incubation of cerebellar neurons with nicked (10-60 nM) or unnicked(7-40 nM) TeNT E234A at 4° C. followed by the addition of 0.2 nM nativetoxin, antagonized the native toxin's inhibition of transmitter releaseat 37° C. to similar extents (FIG. 7).

As demonstrated in FIG. 9, exposure of mouse diaphragm to 100 nM TeNTE234A at 4° C. for 60 minutes prior to adding 1 nM native toxinprolonged the time taken to cause neuromuscular paralysis.

Mouse phrenic-nerve hemi-diaphragm was incubated in KR at 37° C. with 20nM recombinant TeNT E234A (Δ) whilst stimulating the nerve (0.2 Hz,1.5-2.5 v) and recording muscle tension. For assessing competition,hemi-diaphragms were incubated for 60 minutes at 4° C. with MKRcontaining 0.1% BSA only (□), or the latter plus 100 nM nicked TeNTE234A (∘), before the addition of 1 nM native TeNT. Following 30 minutesexposure to the latter, the tissues were washed three times with MKR andtwice with KR. The temperature was raised to 37° C. and the nervestimulated with recording of the evoked muscle twitch, as outlinedabove. This apparent competition for toxin binding by the mutant seenwith both tissues demonstrates that the recombinant di-chain TeNTexhibits much higher affinity for the cell surface receptors than theheavy chain or H_(c) of TeNT alone. These results suggest that theconformation of the recombinant di-chain TeNT has high affinity to thecell surface receptor.

Moreover, and very significantly, these data demonstrate thatrecombinant molecules can be made according to the inventive methods ofthe present patent application having specific binding for the samecellular receptor as TeNT. However, such molecules may, like the E234Amutant, be inactive as toxin molecules but will retain the ability to betaken up by the target cell; thus serving as potential transportermolecules.

Example 8 Expression of Single-Chain BoNT/A

Using methods similar to those described above, DNA fragments containingthe BoNT subtype A neurotoxin H and L chains were ligated together,separated by the EK cleavage site. This single-chain toxin codingsequence was inserted into a variety of expression vectors containingdifferent N terminal sequences and promoters, as shown in Table 4,below. TABLE 4 Tag Size Fusion Size Vector Promoter Fusion Tag (aminoacids) (kDa) E. coli strain pTrcSCPHY trc Poly His 18 150 JM109pCalSCPHY T7 Calmodulin binding 31 154 BL21 (DE3) protein pETSCPHY T7Poly His 32 154 BL21 (DE3) pGEXSCPHY tac Glutathione-S- 224 177 JM109tranferase pMALPHY tac Maltose Binding 390 193 JM109 Protein

The “fusion tags” each comprised a member of a specific binding complexas a purification aid and to improve the solubility and stability of theexpressed protein. These plasmids were transformed into the E. colistrains indicated in Table 4 and expression of the single-chain toxinwas monitored.

In another experiment, the single-chain BoNT/A construct was insertedinto plasmid pMAL-c2 between the Bam HI and Hind III restriction sites,resulting in a coding sequence for a fusion polypeptide containing themaltose binding protein at the N terminus, followed by a Factor Xacleavage site. Transformant JM 109 colonies were selected in Luria brothcontaining ampicillin. Expression was induced by the addition of IPTG toa final concentration of 0.3 mM. As for the TeNT construct, aliquots ofthe cell culture were collected before and after induction, the cells ineach sample lysed by sonication, and the supernatant prepared forSDS-PAGE under both reducing and non-reducing conditions. Followingelectrophoresis to separate the proteins according to apparent molecularweight, the gel was subjected to a Western blot using an antibody raisedagainst the H chain of BoNT/A. The Western blot resulted in theappearance of an immunologically reactive single-chain toxin band ofapparent molecular weight approximately 200 kDa. Further modificationsof the single-chain BoNT molecule to eliminate fortuitous proteasecleavage sites (similar to those modifications made at the TeNT sitelabile to trypsin and Arg C protease, described above) will result ineven greater stability of the single-chain BoNT/A molecule.

Example 9 Construction of a Plasmid Vector Expressing BoNT/E

A plasmid expressing a single-chain recombinant version of theneurotoxin from Clostridium botulinum subtype E (strain Beluga) (BoNT/E)was constructed as follows. PCR primers were designed based on the EMBLdatabase cDNA sequence of the BoNT/E neurotoxin (Genbank accessionnumber X62089) This nucleotide sequence is represented herein as SEQ IDNO: 76. gaattcaagt agtagataat aaaaataatg ccacagattt ttattattaataatgatata tttatctcta actgtttaac tttaacttat aacaatgtaa atatatatttgtctataaaa aatcaagatt acaattgggt tatatgtgat cttaatcatg atataccaaaaaagtcatat ctatggatat taaaaaatat ataaatttaa aattaggaga tgctgtatatgccaaaaatt aatagtttta attataatga tcctgttaat gatagaacaa ttttatatattaaaccaggc ggttgtcaag aattttataa atcatttaat attatgaaaa atatttggataattccagag agaaatgtaa ttggtacaac cccccaagat tttcatccgc ctacttcattaaaaaatgga gatagtagtt attatgaccc taattattta caaagtgatg aagaaaaggatagattttta aaaatagtca caaaaatatt taatagaata aataataatc tttcaggagggattttatta gaagaactgt caaaagctaa tccatattta gggaatgata atactccagataatcaattc catattggtg atgcatcagc agttgagatt aaattctcaa atggtagccaagacatacta ttacctaatg ttattataat gggagcagag cctgatttat ttgaaactaacagttccaat atttctctaa gaaataatta tatgccaagc aatcaccgtt ttggatcaatagctatagta acattctcac ctgaatattc ttttagattt aatgataatt gtatgaatgaatttattcaa gatcctgctc ttacattaat gcatgaatta atacattcat tacatggactatatggggct aaagggatta ctacaaagta tactataaca caaaaacaaa atcccctaataacaaatata agaggtacaa atattgaaga attcttaact tttggaggta ctgatttaaacattattact agtgctcagt ccaatgatat ctatactaat cttctagctg attataaaaaaatagcgtct aaacttagca aagtacaagt atctaatcca ctacttaatc cttataaagatgtttttgaa gcaaagtatg gattagataa agatgctagc ggaatttatt cggtaaatataaacaaattt aatgatattt ttaaaaaatt atacagcttt acggaatttg atttacgaactaaatttcaa gttaaatgta ggcaaactta tattggacag tataaatact tcaaactttcaaacttgtta aatgattcta tttataatat atcagaaggc tataatataa ataatttaaaggtaaatttt agaggacaga atgcaaattt aaatcctaga attattacac caattacaggtagaggacta gtaaaaaaaa tcattagatt ttgtaaaaat attgtttctg taaaaggcataaggaaatca atatgtatcg aaataaataa tggtgagtta ttttttgtgg cttccgagaatagttataat gatgataata taaatactcc taaagaaatt gacgatacag taacttcaaataataattat gaaaatgatt tagatcaggt tattttaaat tttaatagtg aatcagcacctggactttca gatgaaaaat taaatttaac tatccaaaat gatgcttata taccaaaatatgattctaat ggaacaagtg atatagaaca acatgatgtt aatgaactta atgtatttttctatttagat gcacagaaag tgcccgaagg tgaaaataat gtcaatctca cctcttcaattgatacagca ttattagaac aacctaaaat atatacattt ttttcatcag aatttattaataatgtcaat aaacctgtgc aagcagcatt atttgtaagc tggatacaac aagtgttagtagattttact actgaagcta accaaaaaag tactgttgat aaaattgcag atatttctatagttgttcca tatataggtc ttgctttaaa tataggaaat gaagcacaaa aaggaaattttaaagatgca cttgaattat taggagcagg tattttatta gaatttgaac ccgagcttttaattcctaca attttagtat tcacgataaa atctttttta ggttcatctg ataataaaaataaagttatt aaagcaataa ataatgcatt gaaagaaaga gatgaaaaat ggaaagaagtatatagtttt atagtatcga attggatgac taaaattaat acacaattta ataaaagaaaagaacaaatg tatcaagctt tacaaaatca agtaaatgca attaaaacaa taatagaatctaagtataat agttatactt tagaggaaaa aaatgagctt acaaataaat atgatattaagcaaatagaa aatgaactta atcaaaaggt ttctatagca atgaataata tagacaggttcttaactgaa agttctatat cctatttaat gaaaataata aatgaagtaa aaattaataaattaagagaa tatgatgaga atgtcaaaac gtatttattg aattatatta tacaacatggatcaatcttg ggagagagtc agcaagaact aaattctatg gtaactgata ccctaaataatagtattcct tttaagcttt cttcttatac agatgataaa attttaattt catattttaataaattcttt aagagaatta aaagtagttc agttttaaat atgagatata aaaatgataaatacgtagat acttcaggat atgattcaaa tataaatatt aatggagatg tatataaatatccaactaat aaaaatcaat ttggaatata taatgataaa cttagtgaag ttaatatatctcaaaatgat tacattatat atgataataa atataaaaat tttagtatta gtttttgggtaagaattcct aactatgata ataagatagt aaatgttaat aatgaataca ctataataaattgtatgaga gataataatt caggatggaa agtatctctt aatcataatg aaataatttggacattcgaa gataatcgag gaattaatca aaaattagca tttaactatg gtaacgcaaatggtatttct gattatataa ataagtggat ttttgtaact ataactaatg atagattaggagattctaaa ctttatatta atggaaattt aatagatcaa aaatcaattt taaatttaggtaatattcat gttagtgaca atatattatt taaaatagtt aattgtagtt atacaagatatattggtatt agatatttta atatttttga taaagaatta gatgaaacag aaattcaaactttatatagc aatgaaccta atacaaatat tttgaaggat ttttggggaa attatttgctttatgacaaa gaatactatt tattaaatgt gttaaaacca aataacttta ttgataggagaaaagattct actttaagca ttaataatat aagaagcact attcttttag ctaatagattatatagtgga ataaaagtta aaatacaaag agttaataat agtagtacta acgataatcttgttagaaag aatgatcagg tatatattaa ttttgtagcc agcaaaactc acttatttccattatatgct gatacagcta ccacaaataa agagaaaaca ataaaaatat catcatctggcaatagattt aatcaagtag tagttatgaa ttcagtagga aattgtacaa tgaattttaaaaataataat ggaaataata ttgggttgtt aggtttcaag gcagatactg tcgttgctagtacttggtat tatacacata tgagagatca tacaaacagc aatggatgtt tttggaactttatttctgaa gaacatggat ggcaagaaaa ataaaaatta gattaaacgg ctaaagtcat aaattc

The forward primer had the following nucleotide base sequence: (SEQ IDNO: 77) CCCGGATCC CCA AAA ATT AAT AGT TTT AAT TAT AAT G

where the BamHI endonuclease site is underlined and the sequence of thelight chain minus the start codon is in bold. The inverse primer had thesequence: (SEQ ID NO: 78) CCCCTGCAG tca TTT TTC TTG CCA TCC ATG TTC TTC

where the PstI endonuclease site is underlined, the end of the codingregion of the heavy chain is in bold, and the stop codon is in lowercase. These primers were made using standard DNA synthesis methodology.

The two primers were used in a PCR reaction containing different amountsof Clostridium botulinum type E (strain beluga) chromosomal DNA. The PCRreaction employed a DNA polymerase with proofreading activity (Pfx DNApolymerase, obtained from Life Technology) in order to avoid sequenceerrors in the amplified gene. The amplification reaction conditions wereas follows: 30 cycles of: a 45 second denaturation at 95° C., followedby a 45 second annealing step at 56° C., followed by a primer extensionreaction for 3 minutes 48 seconds at 68° C.

The PCR product was digested with BamHI and HindIII, and the digestsubjected to agarose gel electrophoresis. Staining of the agarose gelwith ethidium bromide revealed a major DNA fragment of approximately 3.5kilobases (see FIG. 10). The band containing this fragment was excisedfrom the gel, and the DNA purified from the agarose and ligated to BamHIand HindIII-cut pQE30 vector (Qiagen). The resulting ligated plasmid wasused to transform E. coli strain JM 109 as described above, and thetransformants plated onto selective LB agar plates. Several clones wererecovered and the presence of the correct BoNT/E DNA insert checked byrestriction digest. The resultant construct contains the BoNT/E gene(minus the first methionine) fused to the His₆ tag of the pQE30 vector,and contains 2 extra amino acid residues (glycine, serine), which arecontributed by the engineered BamHI site.

Example 10 Construction of a Proteolytically-Inactive Mutant of BoNT/Eby Site Directed Mutagenesis

By mutating the glutamic acid at position 212 (within the active site)of the BoNT/E polypeptide construct to glutamine, aproteolytically-inactive and non-toxic single-chain BoNT/E polypeptidewas obtained.

The glutamine replacement was introduced on the forward primer usingroutine site directed mutagenesis methods. The mutagenic DNA primer hadthe sequence cagTTAATACATTCATTA CATGGACTATATG (SEQ ID NO: 79), where thecodon encoding glutamine at position 212 is indicated in small letters.An inverse PCR reaction was performed using the above primer, along withthe reverse primer ATGCATTAATGTAAGAGCAGGATCTT (SEQ ID NO: 80) and PfxDNA polymerase (Life Technology) as above. The PCR template was thewild-type single-chain BoNT/E construct (termed pQEESCwt). The cyclingparameters (30 cycles) were as follows: 1) a 45 second denaturation stepat 95° C.; 2) a 45 second annealing step at 56° C.; and 3) a 7 minute 10second extension step at 68° C.

At the end of the amplification reaction, the DNA template was digestedby the restriction enzyme DpnI to permit selection of mutated clonesonly. After subjecting the PCR product to agarose gel electrophoresis, aband of approximately 7 kilobases was removed and the DNA purified andused for self-ligation in the presence of T4 DNA ligase (Promega) andpolynucleotide kinase (Promega) to permit phosphorylation of the PCRproduct. The ligation mixture was used to transform E. coli strainDH10B, and the transformants plated onto selective agar plates. Thepresence of the correct plasmide construct was verified in severalrepresentative transformants by restriction digest and the mutationconfirmed also by DNA sequencing. FIG. 11 shows the protocol forconstruction of the mutant BoNT/E plasmid, and an ethidiumbromide-stained agarose gel of the PCR reaction mixture (lanes 2 and 3)versus molecular weight markers (lane 1).

Example 11 Purification of Single-Chain Recombinant BoNT/E

The presence of the histidine tag at the N-terminus of the expressedprotein allowed a single-step purification of the recombinant neurotoxinby metal-affinity chromatography.

The E. coli strain M15 (Qiagen) was used for expression of the BoNT/Esingle-chain construct. This strain carries an endogenous plasmid(pREP4, kanamycin resistant) containing a region encoding the lac I^(q)repressor gene in order to prevent transcription of the neurotoxin geneprior to induction with IPTG. The pQE30 vector contain a T5bacteriophage RNA polymerase promoter, which is also recognized by E.coli RNA polymerase.

A colony of M15 cells containing pQEESCwt was grown at 37° C. overnightin 5 ml of 2TY medium containing 0.1 mg/ml ampicillin; 0.025 mg/mlkanamycin and 0.2% glucose (w/v), and the resultant culture used toinoculate 500 ml of the same medium. When this second culture reached anoptical density of 0.5-0.8 at 600 nm, IPTG was added to a finalconcentration of 0.3 mM and the culture incubated at 25° C. overnight topermit expression of the neurotoxin.

Subsequent centrifugation of the culture yielded ˜2.3 g of wet cellpellet which was resuspended in 10 ml of extraction buffer (20 mM HepespH 7.0, 300 mM NaCl, 5 mM benzamidine, 2 μM pepstatin and 2 μM E-64).Lysozyme was added to a final concentration of 0.25 mg/ml, and the cellsuspension incubated on ice for 60 minutes. Approximately 0.5 ml ofglass beads (0.1 mm diameter from Biospec) was added to the cellsuspension, followed by vortexing for 2 minutes to break the cells.Cell-free extracts was obtained by centrifugation at 10,000×g for 30minutes at 4° C. The supernatant was incubated with 0.5 ml of Talon®cobalt metal affinity resin (Clontech) pre-washed with extraction bufferin a rocking platform for 45 minutes at 4° C. The resin was then loadedinto a disposable chromatography column and washed twice with 10 bedvolumes of wash buffer (20 mM Hepes pH 7.0, 300 mM NaCl, 2 mM imidazole)before eluting the bound neurotoxin in 6 bed volumes of elution buffer(20 mM Hepes pH 7.0, 300 mM NaCl, 150 mM imidazole).

The elute was dialyzed overnight at 4° C. against 10 mM Hepes (pH 7.0)containing 150 mM NaCl and concentrated by centrifugal filtration (MWcutoff 10 KDa) to a final concentration of 1 mg/ml protein.

As shown in FIG. 12, the purity of the affinity-purified toxin wasdemonstrated by SDS-PAGE under reducing conditions, followed byCoomassie staining and Western-blotting, detecting the N-terminus with amouse monoclonal anti-His antibody from Quiagen (diluted 2000 fold).Enhanced Chemiluminescence solutions (Santa Cruz) and mouse secondaryhorseradish peroxidase (affinity purified from Sigma) were used fordetection of bound antibody. Approximately 2 μg of protein samples wereloaded per well.

Example 12 Trypsin Activation of Purified Recombinant BoNT/ESingle-Chain Polypeptide

Purified BoNT/E single-chain neurotoxin polypeptide samples wereactivated by nicking the single-chain with trypsin (1.5 μg/ml finalconcentration) for 60 minutes at a concentration of 1 mg toxin/ml in 10mm Hepes (pH 7.0), 150 mM NaCl. Following the reaction, the trypsin wasinactivated using 0.5 mM PMSF and 10 μg trypsin inhibitor/ml. Thequality of the trypsinization was assessed and verified by SDS-PAGEunder both reducing and non-reducing conditions, then staining withCoomassie staining and Western blotting the polyacrylamide gel using amouse monoclonal anti-His antibody (Quiagen, diluted 2000-fold) and amouse monoclonal anti-H_(C) IgG (diluted 26-fold). As shown in FIG. 13,the Commassie-stained nicked protein resolves into two bands underreducing conditions, while the heavy and light chains remaindisulfide-linked under non-reducing conditions, similar to the nativetoxin. The antibody-detected recombinant heavy chain is of approximatelyidentical size as its wild-type Clostridium counterpart, whereas therecombinant light chain migrates at a slightly higher molecular weightcompared to the native protein. This latter characteristic is due to theextra residues provided by the His₆ tag at the N-terminus.

Example 13 Recombinant BoNT/E is Proteolytically Active

Stock solutions (1 μM) of native nicked BoNT/E toxin, un-nickedsingle-chain recombinant toxin, nicked di-chain recombinant toxin, andnicked mutant (E212Q) BoNT/E were prepared in HEPES-buffered saline(HBS, 150 mM NaCl, 10 mM HEPES, pH 7.4, 10 μg/ml BSA). These sampleswere incubated for 30 minutes at 37° C. in the absence or presence of 20mM DTT, and then serially diluted in 0.02 ml of HBS to the finalconcentrations shown in FIG. 14.

A recombinant peptide containing amino acids 140-205 of SNAP-25 fused toglutathione-S-transferase (termed GST-SNAP-25 [140-205]) was used as aprotease substrate to test the proteolytic activity of the recombinantBoNT/E polypeptides. Ten micrograms this protease substrate wasincubated with the toxin samples. The digestion reaction was allowed toproceed for 30 minutes at 37° C. in the absence or presence of 2 mM DTT,and stopped by addition of SDS-PAGE sample buffer followed by boilingfor 5 minutes.

The resultant samples were analyzed by SDS-PAGE (3 μg of GST-SNAP-25[140-205] per lane) and silver staining. As FIG. 14 demonstrates, evenunnicked recombinant single-chain toxin retains proteolytic activity. Asexpected, the mutant E212Q BoNT/E construct has no detectableproteolytic activity. FIG. 14 shows only the GST-SNAP-25 [140-205]bands.

Example 14 Nicking Makes Recombinant BoNT/E Fully Functional

Cerebellar neurons maintained for 10 days in culture (2×10⁶/22 mmdiameter well) were washed with Krebs-Ringer HEPES (KRH) buffer, thenexposed to the specified concentrations of BoNT/E native (●),trypsin-nicked recombinant (∘), or un-nicked single-chain (▾) BoNT/E.(See FIG. 15). After 60 minutes at 37° C., the toxin-containing bufferwas removed and the cells were washed twice, then incubated with KRHbuffer containing 0.25 μCi/ml [¹⁴C]-labeled glutamine (i.e. theglutamate precursor). After 45 minutes, the latter medium was removedand the neurons were washed four times at 37° C. prior to assessment oftransmitter glutamate release. Control and toxin-treated neurons wereincubated for 5 minutes at 37° C. in KRH buffer containing either 1.4 mMCa²⁺ or 0.5 mM EGTA to assess Ca²⁺-independent release; aliquots werethen removed for determination of their [¹⁴C]-glutamate content (seebelow).

Immediately after removal of the basal medium, KRH buffer containing 50mM KCl and either 1.4 mM Ca²⁺ or 0.5 mM EGTA was added; as before,aliquots were removed for [¹⁴C]-glutamate assay after a 5 minutestimulation period. Finally, neurons were solubilized with 20 mMEGTA.NaOH pH 7.5 containing 1% (w/v) SDS and aliquots were removed todetermine the amounts of radioactivity remaining within the cells. Theamount of [¹⁴C]-glutamate in each of the samples was assayed byscintillation counting and the levels released under basal andstimulated conditions were expressed as percentages relative to thecalculated total cell content.

The percent [¹⁴C]-glutamate content in the EGTA-containing buffer foreach sample was subtracted from the values recorded in Ca²⁺-containingKRH samples in order to obtain the Ca²⁺-dependent component of release,and the latter basal readings were subtracted from values obtained for50 mM KCl samples to yield K⁺-evoked Ca²⁺-dependent release. The values,thus, obtained from toxin-treated neurons are expressed relative totoxin-free controls.

FIG. 15 shows that, despite retaining proteolytic activity, theun-nicked recombinant BoNT/E has markedly less activity than either thenative BoNT/E or the nicked recombinant version. This finding mayreflect the inability of the un-nicked toxin to adequately enter thetarget cell. Additionally, the nicked recombinant version appears to bemore effective in inhibiting glutamate release than the native toxin.

Example 15 Recombinant BoNT/E has a Neuromuscular Paralytic ActivityEquivalent to that of the Native Toxin at Mouse Neuromuscular EndplatesNicking Increases Potency

Mouse phrenic-nerve hemi-diaphragms were bathed in KR supplemented with0.1% BSA and saturated with 95% O₂/5% CO₂. The phrenic nerves werestimulated (0.2 Hz, 1.5-2.5 mV) and nerve evoked muscle tension wasrecorded before and after the addition of (FIG. 16A) 0.2 nM recombinantnicked BoNT/E (∘) or 0.2 nM native BoNT/E (□), and (FIG. 16B) 1 nMrecombinant un-nicked (∘), 1 nM recombinant nicked (●) or 0.05 nMrecombinant nicked (∇) BoNT/E. As shown in FIGS. 6A and 16B, therecombinant nicked BoNT/E is an effective paralytic agent, displayinggreater activity in this assay that the native toxin. The un-nickedtoxin displays significantly lower activity than the nicked toxin inthis assay.

The neuromuscular paralytic activity of recombinant nicked BoNT/E wasalso demonstrated in mice by intra-muscular injection into hind-limbmuscles. This resulted in paralysis, as assessed by the toe spreadreflex assay, with a pattern of symptoms typical of botulism.

The in vivo neurotoxicity of the nicked, recombinant neurotoxin wasestablished, by injecting the toxin into mice, to have a specificneurotoxicity of less than 10⁷ mouse LD₅₀ units per mg.

Example 16 The BoNT/E E212Q Protease Inactive Mutant AntagonisesBoNT/E-Induced Neuroparalysis

A mouse phrenic-nerve hemi-diaphragm was exposed to 10 nM BoNT/E E212Qin KR medium, the nerve was stimulated and evoked muscle tension wasrecorded. As indicated by FIG. 17, the BoNT E212Q mutant does notinhibit neurotransmission, as determined by its failure to reducenerve-evoked muscle tension (∘). To assess the ability of this non-toxicmutant to antagonise the activity of the native toxin, mousephrenic-nerve hemi-diaphragms were bathed for 60 minutes at 4° C. in MKRsupplemented with 0.1% BSA and saturated with 95% O₂/5% CO₂, without (□)or with (Δ) the inclusion of 5 nM BoNT/E E212Q. Native nicked BoNT/E wasadded to each bath (0.05 nM final) and the tissues were incubated for afurther 30 min. The nerve-muscles were then washed three times each withMKR followed by KR, before the temperature was raised to 37° C., thenerve stimulated and evoked muscle tension recorded.

As shown in FIG. 17, the onset of native BoNT/E activity in this assaywas delayed and antagonized when the phrenic-nerve hemi-diaphragms arepreincubated with the E212Q protease inactive mutant, thereby indicatingthat the recombinant mutant faithfully binds to the same cell surfacereceptor as does the native toxin. Thus, the methods of the presentpatent application can be used to produce recombinant and modifiedtoxins having fully functional receptor binding domains, andBoNT-related transported molecules for the intracellular delivery oftherapeutic agents.

Example 17 Construction of an Activatable Clostridial Toxin Comprisingan Amino-Terminally Presented Binding Element

This example illustrates how to make an activatable Clostridial toxindisclosed in the present specification comprising a binding elementlocated at the amino terminus of the modified toxin.

17a. A Binding Element-Translocation Element-Exogenous Protease CleavageSite-Therapeutic Element Organization.

A polynucleotide molecule based on BoNT/A-TEV-αMSHAP4A (SEQ ID NO: 124)will be synthesized using standard procedures (BlueHeron® Biotechnology,Bothell, Wash.). This polynucleotide molecule encodes a BoNT/A modifiedto replace amino acids 872-1296 of SEQ ID NO: 1, a BoNT/A H_(C) bindingelement, with SEQ ID NO: 81, an αMSH peptide and to incorporate a TEVprotease site of SEQ ID NO: 24 within the di-chain loop region, arrangedin an amino to carboxyl linear organization as depicted in FIG. 20A. TheIn addition, the altered binding element further comprises at its aminoterminus, a PAR 1 leader sequence ending in an enterokinse cleavagesite, which, upon cleavage, results in exposing the first amino acid ofthe αMSH binding element. Oligonucleotides of 20 to 50 bases in lengthare synthesized using standard phosphoramidite synthesis. Theseoligonucleotides will be hybridized into double stranded duplexes thatare ligated together to assemble the full-length polynucleotidemolecule. This polynucleotide molecule will be cloned using standardmolecular biology methods into a pUCBHB1 vector at the SmaI site togenerate pUCBHB1/BoNT/A-TEV-αMSHAP4A. The synthesized polynucleotidemolecule is verified by sequencing using Big Dye Terminator™ Chemistry3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer(Applied Biosystems, Foster City, Calif.).

If desired, an expression optimized polynucleotide molecule based onBoNT/A-TEV-αMSHAP4A can be synthesized in order to improve expression inan Escherichia coli strain. The polynucleotide molecule encoding theBoNT/A-TEV-αMSHAP4A will be modified to 1) contain synonymous codonstypically present in native polynucleotide molecules of an Escherichiacoli strain; 2) contain a G+C content that more closely matches theaverage G+C content of native polynucleotide molecules found in anEscherichia coli strain; 3) reduce polymononucleotide regions foundwithin the polynucleotide molecule; and/or 4) eliminate internalregulatory or structural sites found within the polynucleotide molecule,see, e.g., Lance E. Steward et al., Optimizing Expression of ActiveBotulinum Toxin Type E, International Patent Publication WO 2006/011966(Feb. 2, 2006); Lance E. Steward et al., Optimizing Expression of ActiveBotulinum Toxin Type A, International Patent Publication WO 2006/017749(Feb. 16, 2006). Once sequence optimization is complete,oligonucleotides of 20 to 50 bases in length are synthesized usingstandard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A-TEV-αMSHAP4A.The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.). If so desired, expression optimization to a different organism,such as, e.g., a yeast strain, an insect cell-line or a mammalian cellline, can be done, see, e.g., Steward, supra, (Feb. 2, 2006); andSteward, supra, (Feb. 16, 2006).

A similar cloning strategy will be used to make pUCBHB1 cloningconstructs for BoNT/B-TEV-αMSHAP4A, a modified BoNT/B where amino acids861-1291 of SEQ ID NO: 2 are replaced with SEQ ID NO: 81;BoNT/C1-TEV-αMSHAP4A, a modified BoNT/C1 where amino acids 869-1291 ofSEQ ID NO: 3 are replaced with SEQ ID NO: 81; BoNT/D-TEV-αMSHAP4A, amodified BoNT/D where amino acids 865-1276 of SEQ ID NO: 4 are replacedwith SEQ ID NO: 81; BoNT/E-TEV-αMSHAP4A, a modified BoNT/E where aminoacids 848-1252 of SEQ ID NO: 5 are replaced with SEQ ID NO: 81;BoNT/F-TEV-αMSHAP4A, a modified BoNT/F where amino acids 867-1274 of SEQID NO: 6 are replaced with SEQ ID NO: 81; BoNT/G-TEV-αMSHAP4A, amodified BoNT/G where amino acids 866-1297 of SEQ ID NO: 7 are replacedwith SEQ ID NO: 81; TeNT-TEV-αMSHAP4A, a modified TeNT where amino acids882-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 81;BaNT-TEV-αMSHAP4A, a modified BaNT where amino acids 858-1268 of SEQ IDNO: 9 are replaced with SEQ ID NO: 81; and BuNT-TEV-αMSHAP4A, a modifiedBuNT where amino acids 848-1251 of SEQ ID NO: 10 are replaced with SEQID NO: 81.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-TEV-AP4A that will replace the H_(C) bindingelement from a Clostridial toxin the with an binding element comprising,e.g., a melanocyte stimulating hormone comprising SEQ ID NO: 82 or SEQID NO: 83; an adrenocorticotropin comprising SEQ ID NO: 84 or SEQ ID NO:85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO: 87; aneuropeptide derived from a melanocortin peptide comprising SEQ ID NO:88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO: 91or SEQ ID NO: 92; a chromogranin A peptide comprising SEQ ID NO: 93, SEQID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin B peptide comprising SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105; a chromogranin C peptide comprising SEQ ID NO: 106; a tachykininpeptide comprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118; aNeuropeptide Y related peptide comprising SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin AP4A comprising an exogenous proteasecleavage site incorporated within the di-chain loop region, e.g, abovine enterokinase protease cleavage site comprising SEQ ID NO: 21; aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a Tobacco Vein MottlingVirus protease cleavage site comprising SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3C protease cleavagesite comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisin cleavage sitecomprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51; ahydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53, orSEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ ID NO:56; a non-human Caspase 3 protease cleavage site comprising SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ IDNO: 63.

To construct pET29/BoNT/A-TEV-αMSHAP4A, a pUCBHB1/BoNT/A-TEV-αMSHAP4Aconstruct will be digested with restriction endonucleases that 1) willexcise the polynucleotide molecule encoding the open reading frame ofBoNT/A-TEV-αMSHAP4A; and 2) will enable this polynucleotide molecule tobe operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison,Wis.). This insert will be subcloned using a T4 DNA ligase procedureinto a pET29 vector that is digested with appropriate restrictionendonucleases to yield pET29/BoNT/A-TEV-αMSHAP4A. The ligation mixturewill be transformed into chemically competent E. coli DH5α cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, will beplated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL ofKanamycin, and will be placed in a 37° C. incubator for overnightgrowth. Bacteria containing expression constructs will be identified asKanamycin resistant colonies. Candidate constructs will be isolatedusing an alkaline lysis plasmid mini-preparation procedure and will beanalyzed by restriction endonuclease digest mapping to determine thepresence and orientation of the insert. This cloning strategy will yielda pET29 expression construct comprising the polynucleotide moleculeencoding the BoNT/A-TEV-αMSHAP4A operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide.

A similar cloning strategy will be used to make pET29 expressionconstructs for other modified Clostridial toxin-TEV-αMSHAP4A toxins,such as, e.g., BoNT/B-TEV-αMSHAP4A, BoNT/C1-TEV-αMSHAP4A,BoNT/D-TEV-αMSHAP4A, BoNT/E-TEV-αMSHAP4A, BoNT/F-TEV-αMSHAP4A,BoNT/G-TEV-αMSHAP4A TeNT-TEV-αMSHAP4AB, BaNT-TEV-αMSHAP4A, orBuNT-TEV-αMSHAP4A. Likewise, a similar cloning strategy will be used tomake pET29 expression constructs comprising a polynucleotide moleculeencoding a modified Clostridial toxin-TEV-AP4B comprising a bindingelement such as, e.g, a melanocyte stimulating hormone comprising SEQ IDNO: 82 or SEQ ID NO: 83; an adrenocorticotropin comprising SEQ ID NO: 84or SEQ ID NO: 85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO:87; a neuropeptide derived from a melanocortin peptide comprising SEQ IDNO: 88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO:91 or SEQ ID NO: 92; a chromogranin A peptide comprising SEQ ID NO: 93,SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:98, SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin B peptide comprisingSEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ IDNO: 105; a chromogranin C peptide comprising SEQ ID NO: 106; atachykinin peptide comprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO:118; a Neuropeptide Y related peptide comprising SEQ ID NO: 119, SEQ IDNO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123.

Furthermore, a similar cloning strategy will be used to make pET29expression constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-AP4A comprising an exogenous proteasecleavage site incorporated within the di-chain loop region such as, e.g,a bovine enterokinase protease cleavage site comprising SEQ ID NO: 21; aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a Tobacco Vein MottlingVirus protease cleavage site comprising SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3C protease cleavagesite comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisin cleavage sitecomprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51; ahydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53, orSEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ ID NO:56; a non-human Caspase 3 protease cleavage site comprising SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ IDNO: 63.

17b. A Binding Element-Therapeutic Element-Exogenous Protease CleavageSite-Translocation Element Organization.

A polynucleotide molecule based on BoNT/A-TEV-αMSHAP4B (SEQ ID NO: 125)will be synthesized and cloned into a pUCBHB1 vector as described inExample 17a. This polynucleotide molecule encodes a BoNT/A modified toreplace amino acids 872-1296 of SEQ ID NO: 1, a BoNT/A H_(C) bindingelement, with SEQ ID NO: 81, an αMSH peptide and to incorporate a TEVprotease site of SEQ ID NO: 24 within the di-chain loop region, arrangedin an amino to carboxyl linear organization as depicted in FIG. 20B. Inaddition, the altered binding element further comprises at its aminoterminus, a PAR 1 leader sequence ending in an enterokinse cleavagesite, which upon cleavage, results in exposing the first amino acid ofthe αMSH binding element. If so desired, expression optimization to adifferent organism, such as, e.g., a bacteria, a yeast strain, an insectcell-line or a mammalian cell line, can be done as described above, see,e.g., Steward, supra, (Feb. 2, 2006); and Steward, supra, (Feb. 16,2006).

A similar cloning strategy will be used to make pUCBHB1 cloningconstructs for BoNT/B-TEV-αMSHAP4B, a modified BoNT/B where amino acids861-1291 of SEQ ID NO: 2 are replaced with SEQ ID NO: 81;BoNT/C1-TEV-αMSHAP4B, a modified BoNT/C1 where amino acids 869-1291 ofSEQ ID NO: 3 are replaced with SEQ ID NO: 81; BoNT/D-TEV-αMSHAP4B, amodified BoNT/D where amino acids 865-1276 of SEQ ID NO: 4 are replacedwith SEQ ID NO: 81; BoNT/E-TEV-αMSHAP4B, a modified BoNT/E where aminoacids 848-1252 of SEQ ID NO: 5 are replaced with SEQ ID NO: 81;BoNT/F-TEV-αMSHAP4B, a modified BoNT/F where amino acids 867-1274 of SEQID NO: 6 are replaced with SEQ ID NO: 81; BoNT/G-TEV-αMSHAP4B, amodified BoNT/G where amino acids 866-1297 of SEQ ID NO: 7 are replacedwith SEQ ID NO: 81; TeNT-TEV-αMSHAP4B, a modified TeNT where amino acids882-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 81;BaNT-TEV-αMSHAP4B, a modified BaNT where amino acids 858-1268 of SEQ IDNO: 9 are replaced with SEQ ID NO: 81; and BuNT-TEV-αMSHAP4B, a modifiedBuNT where amino acids 848-1251 of SEQ ID NO: 10 are replaced with SEQID NO: 81.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-TEV-AP4B that will replace the H_(C) bindingelement from a Clostridial toxin the with an binding element comprising,e.g, a melanocyte stimulating hormone comprising SEQ ID NO: 82 or SEQ IDNO: 83; an adrenocorticotropin comprising SEQ ID NO: 84 or SEQ ID NO:85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO: 87; aneuropeptide derived from a melanocortin peptide comprising SEQ ID NO:88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO: 91or SEQ ID NO: 92; a chromogranin A peptide comprising SEQ ID NO: 93, SEQID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin B peptide comprising SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105; a chromogranin C peptide comprising SEQ ID NO: 106; a tachykininpeptide comprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118; aNeuropeptide Y related peptide comprising SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-AP4B comprising an exogenous proteasecleavage site incorporated within the di-chain loop region, e.g, abovine enterokinase protease cleavage site comprising SEQ ID NO: 21; aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a Tobacco Vein MottlingVirus protease cleavage site comprising SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3C protease cleavagesite comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisin cleavage sitecomprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51; ahydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53, orSEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ ID NO:56; a non-human Caspase 3 protease cleavage site comprising SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ IDNO: 63.

To construct pET29/BoNT/A-TEV-αMSHAP4B, a pUCBHB1/BoNT/A-TEV-αMSHAP4Bconstruct will be digested with restriction endonucleases that 1) willexcise the polynucleotide molecule encoding the open reading frame ofBoNT/A-TEV-αMSHAP4B; and 2) will enable this polynucleotide molecule tobe operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison,Wis.). This insert will be subcloned using a T4 DNA ligase procedureinto a pET29 vector that is digested with appropriate restrictionendonucleases to yield pET29/BoNT/A-TEV-αMSHAP4B. The ligation mixturewill be transformed into chemically competent E. coli DH5α cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, will beplated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL ofKanamycin, and will be placed in a 37° C. incubator for overnightgrowth. Bacteria containing expression constructs will be identified asKanamycin resistant colonies. Candidate constructs will be isolatedusing an alkaline lysis plasmid mini-preparation procedure and will beanalyzed by restriction endonuclease digest mapping to determine thepresence and orientation of the insert. This cloning strategy will yielda pET29 expression construct comprising the polynucleotide moleculeencoding the BoNT/A-TEV-αMSHAP4B operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide.

A similar cloning strategy will be used to make pET29 expressionconstructs for other modified Clostridial toxin-TEV-αMSHAP4B toxins,such as, e.g., BoNT/B-TEV-αMSHAP4B, BoNT/C1-TEV-αMSHAP4B,BoNT/D-TEV-αMSHAP4B, BoNT/E-TEV-αMSHAP4B, BoNT/F-TEV-αMSHAP4B,BoNT/G-TEV-αMSHAP4B, TeNT-TEV-αMSHAP4B, BaNT-TEV-αMSHAP4B, orBuNT-TEV-αMSHAP4B. Likewise, a similar cloning strategy will be used tomake pET29 expression constructs comprising a polynucleotide moleculeencoding a modified Clostridial toxin-TEV-AP4B comprising a bindingelement such as, e.g, a melanocyte stimulating hormone comprising SEQ IDNO: 82 or SEQ ID NO: 83; an adrenocorticotropin comprising SEQ ID NO: 84or SEQ ID NO: 85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO:87; a neuropeptide derived from a melanocortin peptide comprising SEQ IDNO: 88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO:91 or SEQ ID NO: 92; a chromogranin A peptide comprising SEQ ID NO: 93,SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:98, SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin B peptide comprisingSEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ IDNO: 105; a chromogranin C peptide comprising SEQ ID NO: 106; atachykinin peptide comprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO:118; a Neuropeptide Y related peptide comprising SEQ ID NO: 119, SEQ IDNO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123.

Furthermore, a similar cloning strategy will be used to make pET29expression constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-AP4B comprising an exogenous proteasecleavage site incorporated within the di-chain loop region such as, e.g,a bovine enterokinase protease cleavage site comprising SEQ ID NO: 21; aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a Tobacco Vein MottlingVirus protease cleavage site comprising SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3C protease cleavagesite comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisin cleavage sitecomprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51; ahydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53, orSEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ ID NO:56; a non-human Caspase 3 protease cleavage site comprising SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ IDNO: 63.

Example 18 Construction of an Activatable Clostridial Toxin Comprising aCentrally Presented Altered Targeting Domain

This example illustrates how to make an activatable Clostridial toxindisclosed in the present specification comprising a binding elementlocated between two other domains of the modified toxin.

18a. A Therapeutic Element-Exogenous Protease Cleavage Site-BindingElement-Translocation Element Organization.

A polynucleotide molecule based on BoNT/A-ENT-NPYCP5A (SEQ ID NO: 126)will be synthesized and cloned into a pUCBHB1 vector as described inExample 17a. This polynucleotide molecule encodes a BoNT/A modified toreplace amino acids 872-1296 of SEQ ID NO: 1, a BoNT/A H_(C) bindingelement, with SEQ ID NO: 119, a NPY peptide and to incorporate a bovineenterokinse protease site of SEQ ID NO: 21 within the di-chain loopregion, arranged in an amino to carboxyl linear organization as depictedin FIG. 21A. Cleavage of an enterokinse cleavage site used to form thedi-chain toxin also exposes the first amino acid of the NPY bindingelement. If so desired, expression optimization to a different organism,such as, e.g., a bacteria, a yeast strain, an insect cell-line or amammalian cell line, can be done as described above, see, e.g., Steward,supra, (Feb. 2, 2006); and Steward, supra, (Feb. 16, 2006).

A similar cloning strategy will be used to make pUCBHB1 cloningconstructs for BoNT/B-ENT-NPYCP5A, a modified BoNT/B where amino acids861-1291 of SEQ ID NO: 2 are replaced with SEQ ID NO: 119;BoNT/C1-ENT-NPYCP5A, a modified BoNT/C1 where amino acids 869-1291 ofSEQ ID NO: 3 are replaced with SEQ ID NO: 119; BoNT/D-ENT-NPYCP5A, amodified BoNT/D where amino acids 865-1276 of SEQ ID NO: 4 are replacedwith SEQ ID NO: 119; BoNT/E-ENT-NPYCP5A, a modified BoNT/E where aminoacids 848-1252 of SEQ ID NO: 5 are replaced with SEQ ID NO: 119;BoNT/F-ENT-NPYCP5A, a modified BoNT/F where amino acids 867-1274 of SEQID NO: 6 are replaced with SEQ ID NO: 119; BoNT/G-ENT-NPYCP5A, amodified BoNT/G where amino acids 866-1297 of SEQ ID NO: 7 are replacedwith SEQ ID NO: 119; TeNT-ENT-NPYCP5A, a modified TeNT where amino acids882-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 119;BaNT-ENT-NPYCP5A, a modified BaNT where amino acids 858-1268 of SEQ IDNO: 9 are replaced with SEQ ID NO: 119; and BuNT-ENT-NPYCP5A, a modifiedBuNT where amino acids 848-1251 of SEQ ID NO: 10 are replaced with SEQID NO: 119.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-ENT-CP5A that will replace the H_(C) bindingelement from a Clostridial toxin the with an binding element comprising,e.g, a melanocyte stimulating hormone comprising SEQ ID NO: 81, SEQ IDNO: 82 or SEQ ID NO: 83; an adrenocorticotropin comprising SEQ ID NO: 84or SEQ ID NO: 85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO:87; a neuropeptide derived from a melanocortin peptide comprising SEQ IDNO: 88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO:91 or SEQ ID NO: 92; a chromogranin A peptide comprising SEQ ID NO: 93,SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:98, SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin B peptide comprisingSEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ IDNO: 105; a chromogranin C peptide comprising SEQ ID NO: 106; atachykinin peptide comprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO:118; a Neuropeptide Y related peptide comprising SEQ ID NO: 120, SEQ IDNO: 121, SEQ ID NO: 122 or SEQ ID NO: 123.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-CP5A comprising an exogenous proteasecleavage site incorporated within the di-chain loop region, e.g, aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a TobaccoVein Mottling Virus protease cleavage site comprising SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3Cprotease cleavage site comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisincleavage site comprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51;a hydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53,or SEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ IDNO: 56; a non-human Caspase 3 protease cleavage site comprising SEQ IDNO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 orSEQ ID NO: 63. In addition, a similar cloning strategy will be used tomake pUCBHB1 cloning constructs comprising a polynucleotide moleculeencoding a modified Clostridial toxin-CP5A comprising an exogenousprotease cleavage site incorporated within the di-chain loop region,cleavage of which converts the single-chain polypeptide of the toxininto its di-chain form and also exposes the first amino acid of thebinding element.

To construct pET29/BoNT/A-ENT-NPYCP5A, a pUCBHB1/BoNT/A-ENT-NPYCP5Aconstruct will be digested with restriction endonucleases that 1) willexcise the polynucleotide molecule encoding the open reading frame ofBoNT/A-ENT-NPYCP5A; and 2) will enable this polynucleotide molecule tobe operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison,Wis.). This insert will be subcloned using a T4 DNA ligase procedureinto a pET29 vector that is digested with appropriate restrictionendonucleases to yield pET29/BoNT/A-ENT-NPYCP5A. The ligation mixturewill be transformed into chemically competent E. coli DH5α cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, will beplated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL ofKanamycin, and will be placed in a 37° C. incubator for overnightgrowth. Bacteria containing expression constructs will be identified asKanamycin resistant colonies. Candidate constructs will be isolatedusing an alkaline lysis plasmid mini-preparation procedure and will beanalyzed by restriction endonuclease digest mapping to determine thepresence and orientation of the insert. This cloning strategy will yielda pET29 expression construct comprising the polynucleotide moleculeencoding the BoNT/A-TEV-NPYCP5A operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide.

A similar cloning strategy will be used to make pET29 expressionconstructs for other modified Clostridial toxin-ENT-NPYCP5A toxins, suchas, e.g., BoNT/B-ENT-NPYCP5A, BoNT/C1-ENT-NPYCP5A, BoNT/D-ENT-NPYCP5A,BoNT/E-ENT-NPYCP5A, BoNT/F-ENT-NPYCP5A, BoNT/G-ENT-NPYCP5A,TeNT-ENT-NPYCP5A, BaNT-ENT-NPYCP5A, or BuNT-ENT-NPYCP5A. Likewise, asimilar cloning strategy will be used to make pET29 expressionconstructs comprising a polynucleotide molecule encoding a modifiedClostridial toxin-ENT-CP5B comprising a binding element such as, e.g, amelanocyte stimulating hormone comprising SEQ ID NO: 81, SEQ ID NO: 82or SEQ ID NO: 83; an adrenocorticotropin comprising SEQ ID NO: 84 or SEQID NO: 85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO: 87; aneuropeptide derived from a melanocortin peptide comprising SEQ ID NO:88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO: 91or SEQ ID NO: 92; a chromogranin A peptide comprising SEQ ID NO: 93, SEQID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin B peptide comprising SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105; a chromogranin C peptide comprising SEQ ID NO: 106; a tachykininpeptide comprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118; aNeuropeptide Y related peptide comprising SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122 or SEQ ID NO: 123. If required for function, theselected binding element will be engineered to expose the free aminoterminal amino acid of the binding element.

Furthermore, a similar cloning strategy will be used to make pET29expression constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-CP5A comprising an exogenous proteasecleavage site incorporated within the di-chain loop region such as, e.g,a Tobacco Etch Virus protease cleavage site comprising SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; aTobacco Vein Mottling Virus protease cleavage site comprising SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus3C protease cleavage site comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisincleavage site comprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51;a hydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53,or SEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ IDNO: 56; a non-human Caspase 3 protease cleavage site comprising SEQ IDNO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 orSEQ ID NO: 63. In addition, a similar cloning strategy will be used tomake pET29 expression constructs comprising a polynucleotide moleculeencoding a modified Clostridial toxin-CP5A comprising an exogenousprotease cleavage site incorporated within the di-chain loop region suchas, e.g, an exogenous protease cleavage site which upon cleavageconverts the single-chain polypeptide of the toxin into its di-chainform and also exposes the first amino acid of the binding element.

18b. A Translocation Element-Exogenous Protease Cleavage Site-BindingElement-Therapeutic Element Organization.

A polynucleotide molecule based on BoNT/A-ENT-NPYCP5B (SEQ ID NO: 127)will be synthesized and cloned into a pUCBHB1 vector as described inExample 17a. This polynucleotide molecule encodes a BoNT/A modified toreplace amino acids 872-1296 of SEQ ID NO: 1, a BoNT/A H_(C) bindingelement, with SEQ ID NO: 119, a NPY peptide and to incorporate a bovineenterokinse protease site of SEQ ID NO: 21 within the di-chain loopregion, arranged in an amino to carboxyl linear organization as depictedin FIG. 21B. Cleavage of an enterokinse cleavage site used to form thedi-chain toxin also exposes the first amino acid of the NPY bindingelement. If so desired, expression optimization to a different organism,such as, e.g., a bacteria, a yeast strain, an insect cell-line or amammalian cell line, can be done as described above, see, e.g., Steward,supra, (Feb. 2, 2006); and Steward, supra, (Feb. 16, 2006).

A similar cloning strategy will be used to make pUCBHB1 cloningconstructs for BoNT/B-ENT-NPYCP5B, a modified BoNT/B where amino acids861-1291 of SEQ ID NO: 2 are replaced with SEQ ID NO: 119;BoNT/C1-ENT-NPYCP5B, a modified BoNT/C1 where amino acids 869-1291 ofSEQ ID NO: 3 are replaced with SEQ ID NO: 119; BoNT/D-ENT-NPYCP5B, amodified BoNT/D where amino acids 865-1276 of SEQ ID NO: 4 are replacedwith SEQ ID NO: 119; BoNT/E-ENT-NPYCP5B, a modified BoNT/E where aminoacids 848-1252 of SEQ ID NO: 5 are replaced with SEQ ID NO: 119;BoNT/F-ENT-NPYCP5B, a modified BoNT/F where amino acids 867-1274 of SEQID NO: 6 are replaced with SEQ ID NO: 119; BoNT/G-ENT-NPYCP5B, amodified BoNT/G where amino acids 866-1297 of SEQ ID NO: 7 are replacedwith SEQ ID NO: 119; TeNT-ENT-NPYCP5B, a modified TeNT where amino acids882-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 119;BaNT-ENT-NPYCP5B, a modified BaNT where amino acids 858-1268 of SEQ IDNO: 9 are replaced with SEQ ID NO: 119; and BuNT-ENT-NPYCP5B, a modifiedBuNT where amino acids 848-1251 of SEQ ID NO: 10 are replaced with SEQID NO: 119.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-ENT-CP5B that will replace the H_(C) bindingelement from a Clostridial toxin the with an binding element comprising,e.g, a melanocyte stimulating hormone comprising SEQ ID NO: 81, SEQ IDNO: 82 or SEQ ID NO: 83; an adrenocorticotropin comprising SEQ ID NO: 84or SEQ ID NO: 85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO:87; a neuropeptide derived from a melanocortin peptide comprising SEQ IDNO: 88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO:91 or SEQ ID NO: 92; a chromogranin A peptide comprising SEQ ID NO: 93,SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:98, SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin B peptide comprisingSEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ IDNO: 105; a chromogranin C peptide comprising SEQ ID NO: 106; atachykinin peptide comprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO:118; a Neuropeptide Y related peptide comprising SEQ ID NO: 120, SEQ IDNO: 121, SEQ ID NO: 122 or SEQ ID NO: 123.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-CP5B comprising an exogenous proteasecleavage site incorporated within the di-chain loop region, e.g, aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a TobaccoVein Mottling Virus protease cleavage site comprising SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3Cprotease cleavage site comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisincleavage site comprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51;a hydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53,or SEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ IDNO: 56; a non-human Caspase 3 protease cleavage site comprising SEQ IDNO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 orSEQ ID NO: 63. In addition, a similar cloning strategy will be used tomake pUCBHB1 cloning constructs comprising a polynucleotide moleculeencoding a modified Clostridial toxin-CP5B comprising an exogenousprotease cleavage site incorporated within the di-chain loop region,cleavage of which converts the single-chain polypeptide of the toxininto its di-chain form and also exposes the first amino acid of thebinding element.

To construct pET29/BoNT/A-ENT-NPYCP5B, a pUCBHB1/BoNT/A-ENT-NPYCP5Bconstruct will be digested with restriction endonucleases that 1) willexcise the polynucleotide molecule encoding the open reading frame ofBoNT/A-ENT-NPYCP5B; and 2) will enable this polynucleotide molecule tobe operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison,Wis.). This insert will be subcloned using a T4 DNA ligase procedureinto a pET29 vector that is digested with appropriate restrictionendonucleases to yield pET29/BoNT/A-ENT-NPYCP5B. The ligation mixturewill be transformed into chemically competent E. coli DH5α cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, will beplated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL ofKanamycin, and will be placed in a 37° C. incubator for overnightgrowth. Bacteria containing expression constructs will be identified asKanamycin resistant colonies. Candidate constructs will be isolatedusing an alkaline lysis plasmid mini-preparation procedure and will beanalyzed by restriction endonuclease digest mapping to determine thepresence and orientation of the insert. This cloning strategy will yielda pET29 expression construct comprising the polynucleotide moleculeencoding the BoNT/A-ENT-NPYCP5B operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide.

A similar cloning strategy will be used to make pET29 expressionconstructs for other modified Clostridial toxin-ENT-NPYCP5B toxins, suchas, e.g., BoNT/B-ENT-NPYCP5B, BoNT/C1-ENT-NPYCP5B, BoNT/D-ENT-NPYCP5B,BoNT/E-ENT-NPYCP5B, BoNT/F-ENT-NPYCP5B, BoNT/G-ENT-NPYCP5B,TeNT-ENT-NPYCP5B, BaNT-ENT-NPYCP5B, or BuNT-ENT-NPYCP5B. Likewise, asimilar cloning strategy will be used to make pET29 expressionconstructs comprising a polynucleotide molecule encoding a modifiedClostridial toxin-ENT-CP5B comprising a binding element such as, e.g, amelanocyte stimulating hormone comprising SEQ ID NO: 81, SEQ ID NO: 82or SEQ ID NO: 83; an adrenocorticotropin comprising SEQ ID NO: 84 or SEQID NO: 85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO: 87; aneuropeptide derived from a melanocortin peptide comprising SEQ ID NO:88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO: 91or SEQ ID NO: 92; a chromogranin A peptide comprising SEQ ID NO: 93, SEQID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin B peptide comprising SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105; a chromogranin C peptide comprising SEQ ID NO: 106; a tachykininpeptide comprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118; aNeuropeptide Y related peptide comprising SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122 or SEQ ID NO: 123. If required for function, theselected binding element will be engineered to expose the free aminoterminal amino acid of the binding element.

Furthermore, a similar cloning strategy will be used to make pET29expression constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-CP5B comprising an exogenous proteasecleavage site incorporated within the di-chain loop region such as, e.g,a Tobacco Etch Virus protease cleavage site comprising SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; aTobacco Vein Mottling Virus protease cleavage site comprising SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus3C protease cleavage site comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisincleavage site comprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51;a hydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53,or SEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ IDNO: 56; a non-human Caspase 3 protease cleavage site comprising SEQ IDNO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 orSEQ ID NO: 63. In addition, a similar cloning strategy will be used tomake pET29 expression constructs comprising a polynucleotide moleculeencoding a modified Clostridial toxin-CP5B comprising an exogenousprotease cleavage site incorporated within the di-chain loop region suchas, e.g, an exogenous protease cleavage site which upon cleavageconverts the single-chain polypeptide of the toxin into its di-chainform and also exposes the first amino acid of the binding element.

Example 19 Construction of an Activatable Clostridial Toxin Comprising aCarboxyl-Terminally Presented Altered Targeting Domain

This example illustrates how to make an activatable Clostridial toxindisclosed in the present specification comprising a binding elementlocated at the carboxyl terminus of the modified toxin.

19a. A Therapeutic Element-Exogenous Pro Tease CleavageSite-Translocation Element-Binding Element Organization.

A polynucleotide molecule based on BoNT/A-TEV-GalaninXP6A (SEQ ID NO:128) will be synthesized and cloned into a pUCBHB1 vector as describedin Example 17a. This polynucleotide molecule encodes a BoNT/A modifiedto replace amino acids 872-1296 of SEQ ID NO: 1, a BoNT/A H_(C) bindingelement, with SEQ ID NO: 91, a Galanin peptide and to incorporate a TEVprotease site of SEQ ID NO: 24 within the di-chain loop region, arrangedin an amino to carboxyl linear organization as depicted in FIG. 22A. Ifso desired, expression optimization to a different organism, such as,e.g., a bacteria, a yeast strain, an insect cell-line or a mammaliancell line, can be done as described above, see, e.g., Steward, supra,(Feb. 2, 2006); and Steward, supra, (Feb. 16, 2006).

A similar cloning strategy will be used to make pUCBHB1 cloningconstructs for BoNT/B-TEV-GalaninXP6A, a modified BoNT/B where aminoacids 861-1291 of SEQ ID NO: 2 are replaced with SEQ ID NO: 91;BoNT/C1-TEV-GalaninXP6A, a modified BoNT/C1 where amino acids 869-1291of SEQ ID NO: 3 are replaced with SEQ ID NO: 91; BoNT/D-TEV-GalaninXP6A,a modified BoNT/D where amino acids 865-1276 of SEQ ID NO: 4 arereplaced with SEQ ID NO: 91; BoNT/E-TEV-GalaninXP6A, a modified BoNT/Ewhere amino acids 848-1252 of SEQ ID NO: 5 are replaced with SEQ ID NO:91; BoNT/F-TEV-GalaninXP6A, a modified BoNT/F where amino acids 867-1274of SEQ ID NO: 6 are replaced with SEQ ID NO: 91; BoNT/G-TEV-GalaninXP6A,a modified BoNT/G where amino acids 866-1297 of SEQ ID NO: 7 arereplaced with SEQ ID NO: 91; TeNT-TEV-GalaninXP6A, a modified TeNT whereamino acids 882-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 91;BaNT-TEV-GalaninXP6A, a modified BaNT where amino acids 858-1268 of SEQID NO: 9 are replaced with SEQ ID NO: 91; and BuNT-TEV-GalaninXP6A, amodified BuNT where amino acids 848-1251 of SEQ ID NO: 10 are replacedwith SEQ ID NO: 91.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-TEV-XP6A that will replace the H_(C) bindingelement from a Clostridial toxin the with an binding element comprising,e.g, a melanocyte stimulating hormone comprising SEQ ID NO: 81, SEQ IDNO: 82 or SEQ ID NO: 83; an adrenocorticotropin comprising SEQ ID NO: 84or SEQ ID NO: 85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO:87; a neuropeptide derived from a melanocortin peptide comprising SEQ IDNO: 88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO:92; a chromogranin A peptide comprising SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99 or SEQ ID NO: 100; a chromogranin B peptide comprising SEQ ID NO:101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105; achromogranin C peptide comprising SEQ ID NO: 106; a tachykinin peptidecomprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118; aNeuropeptide Y related peptide comprising SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-XP6A comprising an exogenous proteasecleavage site incorporated within the di-chain loop region, e.g, abovine enterokinase protease cleavage site comprising SEQ ID NO: 21; aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a Tobacco Vein MottlingVirus protease cleavage site comprising SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3C protease cleavagesite comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisin cleavage sitecomprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51; ahydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53, orSEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ ID NO:56; a non-human Caspase 3 protease cleavage site comprising SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ IDNO: 63.

To construct pET29/BoNT/A-TEV-GalaninXP6A, apUCBHB1/BoNT/A-TEV-GalaninXP6A construct will be digested withrestriction endonucleases that 1) will excise the polynucleotidemolecule encoding the open reading frame of BoNT/A-TEV-GalaninXP6A; and2) will enable this polynucleotide molecule to be operably-linked to apET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert willbe subcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/BoNT/A-TEV-GalaninXP6A. The ligation mixture will be transformedinto chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, will be plated on 1.5% Luria-Bertaniagar plates (pH 7.0) containing 50 μg/mL of Kanamycin, and will beplaced in a 37° C. incubator for overnight growth. Bacteria containingexpression constructs will be identified as Kanamycin resistantcolonies. Candidate constructs will be isolated using an alkaline lysisplasmid mini-preparation procedure and will be analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe insert. This cloning strategy will yield a pET29 expressionconstruct comprising the polynucleotide molecule encoding theBoNT/A-TEV-GalaninXP6A operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide.

A similar cloning strategy will be used to make pET29 expressionconstructs for other modified Clostridial toxin-TEV-GalaninXP6A toxins,such as, e.g., BoNT/B-TEV-GalaninXP6A, BoNT/C1-TEV-GalaninXP6A,BoNT/D-TEV-GalaninXP6A, BoNT/E-TEV-GalaninXP6A, BoNT/F-TEV-GalaninXP6A,BoNT/G-TEV-GalaninXP6A, TeNT-TEV-GalaninXP6A, BaNT-TEV-GalaninXP6A, orBuNT-TEV-GalaninXP6A. Likewise, a similar cloning strategy will be usedto make pET29 expression constructs comprising a polynucleotide moleculeencoding a modified Clostridial toxin-TEV-XP6A comprising a bindingelement such as, e.g, a melanocyte stimulating hormone comprising SEQ IDNO: 81, SEQ ID NO: 82 or SEQ ID NO: 83; an adrenocorticotropincomprising SEQ ID NO: 84 or SEQ ID NO: 85; a lipotropin comprising SEQID NO: 86 or SEQ ID NO: 87; a neuropeptide derived from a melanocortinpeptide comprising SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90; agalanin comprising SEQ ID NO: 92; a chromogranin A peptide comprisingSEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin Bpeptide comprising SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104 or SEQ ID NO: 105; a chromogranin C peptide comprising SEQ IDNO: 106; a tachykinin peptide comprising SEQ ID NO: 107, SEQ ID NO: 108,SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ IDNO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117or SEQ ID NO: 118; a Neuropeptide Y related peptide comprising SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123.

Furthermore, a similar cloning strategy will be used to make pET29expression constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-XP6A comprising an exogenous proteasecleavage site incorporated within the di-chain loop region such as, e.g,a bovine enterokinase protease cleavage site comprising SEQ ID NO: 21; aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a Tobacco Vein MottlingVirus protease cleavage site comprising SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3C protease cleavagesite comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisin cleavage sitecomprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51; ahydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53, orSEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ ID NO:56; a non-human Caspase 3 protease cleavage site comprising SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ IDNO: 63.

19b. A Translocation Element-Exogenous Protease CleavageSite-Therapeutic Element-Binding Element Organization.

A polynucleotide molecule based on BoNT/A-TEV-GalaninXP6B (SEQ ID NO:129) will be synthesized and cloned into a pUCBHB1 vector as describedin Example 17a. This polynucleotide molecule encodes a BoNT/A modifiedto replace amino acids 872-1296 of SEQ ID NO: 1, a BoNT/A H_(C) bindingelement, with SEQ ID NO: 91, a Galanin peptide and to incorporate a TEVprotease site of SEQ ID NO: 24 within the di-chain loop region, arrangedin an amino to carboxyl linear organization as depicted in FIG. 22B. Ifso desired, expression optimization to a different organism, such as,e.g., a bacteria, a yeast strain, an insect cell-line or a mammaliancell line, can be done as described above, see, e.g., Steward, supra,(Feb. 2, 2006); and Steward, supra, (Feb. 16, 2006).

A similar cloning strategy will be used to make pUCBHB1 cloningconstructs for BoNT/B-TEV-GalaninXP6B, a modified BoNT/B where aminoacids 861-1291 of SEQ ID NO: 2 are replaced with SEQ ID NO: 91;BoNT/C1-TEV-GalaninXP6B, a modified BoNT/C1 where amino acids 869-1291of SEQ ID NO: 3 are replaced with SEQ ID NO: 91; BoNT/D-TEV-GalaninXP6B,a modified BoNT/D where amino acids 865-1276 of SEQ ID NO: 4 arereplaced with SEQ ID NO: 91; BoNT/E-TEV-GalaninXP6B, a modified BoNT/Ewhere amino acids 848-1252 of SEQ ID NO: 5 are replaced with SEQ ID NO:91; BoNT/F-TEV-GalaninXP6B, a modified BoNT/F where amino acids 867-1274of SEQ ID NO: 6 are replaced with SEQ ID NO: 91; BoNT/G-TEV-GalaninXP6B,a modified BoNT/G where amino acids 866-1297 of SEQ ID NO: 7 arereplaced with SEQ ID NO: 91; TeNT-TEV-GalaninXP6B, a modified TeNT whereamino acids 882-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 91;BaNT-TEV-GalaninXP6B, a modified BaNT where amino acids 858-1268 of SEQID NO: 9 are replaced with SEQ ID NO: 91; and BuNT-TEV-GalaninXP6B, amodified BuNT where amino acids 848-1251 of SEQ ID NO: 10 are replacedwith SEQ ID NO: 91.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-TEV-XP6B that will replace the H_(C) bindingelement from a Clostridial toxin the with an binding element comprising,e.g, a melanocyte stimulating hormone comprising SEQ ID NO: 81, SEQ IDNO: 82 or SEQ ID NO: 83; an adrenocorticotropin comprising SEQ ID NO: 84or SEQ ID NO: 85; a lipotropin comprising SEQ ID NO: 86 or SEQ ID NO:87; a neuropeptide derived from a melanocortin peptide comprising SEQ IDNO: 88, SEQ ID NO: 89 or SEQ ID NO: 90; a galanin comprising SEQ ID NO:92; a chromogranin A peptide comprising SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99 or SEQ ID NO: 100; a chromogranin B peptide comprising SEQ ID NO:101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105; achromogranin C peptide comprising SEQ ID NO: 106; a tachykinin peptidecomprising SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118; aNeuropeptide Y related peptide comprising SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO: 123.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-XP6B comprising an exogenous proteasecleavage site incorporated within the di-chain loop region, e.g, abovine enterokinase protease cleavage site comprising SEQ ID NO: 21; aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a Tobacco Vein MottlingVirus protease cleavage site comprising SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3C protease cleavagesite comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisin cleavage sitecomprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51; ahydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53, orSEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ ID NO:56; a non-human Caspase 3 protease cleavage site comprising SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ IDNO: 63.

To construct pET29/BoNT/A-TEV-αMSHAP4B, a pUCBHB1/BoNT/A-TEV-GalaninXP6Bconstruct will be digested with restriction endonucleases that 1) willexcise the polynucleotide molecule encoding the open reading frame ofBoNT/A-TEV-GalaninXP6B; and 2) will enable this polynucleotide moleculeto be operably-linked to a pET29 vector (EMD Biosciences-Novagen,Madison, Wis.). This insert will be subcloned using a T4 DNA ligaseprocedure into a pET29 vector that is digested with appropriaterestriction endonucleases to yield pET29/BoNT/A-TEV-GalaninXP6B. Theligation mixture will be transformed into chemically competent E. coliDH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shockmethod, will be plated on 1.5% Luria-Bertani agar plates (pH 7.0)containing 50 μg/mL of Kanamycin, and will be placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs will be identified as Kanamycin resistant colonies. Candidateconstructs will be isolated using an alkaline lysis plasmidmini-preparation procedure and will be analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe insert. This cloning strategy will yield a pET29 expressionconstruct comprising the polynucleotide molecule encoding theBoNT/A-TEV-GalaninXP6B operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide.

A similar cloning strategy will be used to make pET29 expressionconstructs for other modified Clostridial toxin-TEV-GalaninXP6B toxins,such as, e.g., BoNT/B-TEV-GalaninXP6B, BoNT/C1-TEV-GalaninXP6B,BoNT/D-TEV-GalaninXP6B, BoNT/E-TEV-GalaninXP6B, BoNT/F-TEV-GalaninXP6B,BoNT/G-TEV-GalaninXP6B, TeNT-TEV-GalaninXP6B, BaNT-TEV-GalaninXP6B, orBuNT-TEV-GalaninXP6B. Likewise, a similar cloning strategy will be usedto make pET29 expression constructs comprising a polynucleotide moleculeencoding a modified Clostridial toxin-TEV-XP6B comprising a bindingelement such as, e.g, a melanocyte stimulating hormone comprising SEQ IDNO: 81, SEQ ID NO: 82 or SEQ ID NO: 83; an adrenocorticotropincomprising SEQ ID NO: 84 or SEQ ID NO: 85; a lipotropin comprising SEQID NO: 86 or SEQ ID NO: 87; a neuropeptide derived from a melanocortinpeptide comprising SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90; agalanin comprising SEQ ID NO: 92; a chromogranin A peptide comprisingSEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100; a chromogranin Bpeptide comprising SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104 or SEQ ID NO: 105; a chromogranin C peptide comprising SEQ IDNO: 106; a tachykinin peptide comprising SEQ ID NO: 107, SEQ ID NO: 108,SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ IDNO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117or SEQ ID NO: 118; a Neuropeptide Y related peptide comprising SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:123.

Furthermore, a similar cloning strategy will be used to make pET29expression constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-XP6B comprising an exogenous proteasecleavage site incorporated within the di-chain loop region such as, e.g,a bovine enterokinase protease cleavage site comprising SEQ ID NO: 21; aTobacco Etch Virus protease cleavage site comprising SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33; a Tobacco Vein MottlingVirus protease cleavage site comprising SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, or SEQ ID NO: 39; a human rhinovirus 3C protease cleavagesite comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45 or SEQ ID NO: 46; a subtilisin cleavage sitecomprising SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51; ahydroxylamine cleavage site comprising SEQ ID NO: 52, SEQ ID NO: 53, orSEQ ID NO: 54; a SUMO/ULP-1 protease cleavage site comprising SEQ ID NO:56; a non-human Caspase 3 protease cleavage site comprising SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ IDNO: 63.

Example 20 Expression of Activatable Clostridial Toxins in a BacterialCell

The following example illustrates a procedure useful for expressing anyof the activatable Clostridial toxins disclosed in the presentspecification in a bacterial cell.

An expression construct, such as, e.g., any of the expression constructsin Examples 17-19, will be introduced into chemically competent E. coliBL21 (DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat-shocktransformation protocol. The heat-shock reaction will be plated onto1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycinand will be placed in a 37° C. incubator for overnight growth.Kanamycin-resistant colonies of transformed E. coli containing theexpression construct will be used to inoculate a baffled flaskcontaining 3.0 mL of PA-0.5G media containing 50 μg/mL of Kanamycinwhich will then placed in a 37° C. incubator, shaking at 250 rpm, forovernight growth. The resulting overnight starter culture will be usedto inoculate a 3 L baffled flask containing ZYP-5052 autoinducing mediacontaining 50 μg/mL of Kanamycin at a dilution of 1:1000. Culturevolumes will range from about 600 mL (20% flask volume) to about 750 mL(25% flask volume). These cultures will be grown in a 37° C. incubatorshaking at 250 rpm for approximately 5.5 hours and will be thentransferred to a 16° C. incubator shaking at 250 rpm for overnightexpression. Cells will be harvested by centrifugation (4,000 rpm at 4°C. for 20-30 minutes) and will be used immediately, or will be storeddry at −80° C. until needed.

Example 21 Purification and Quantification of Activatable ClostridialToxins

The following example illustrates methods useful for purification andquantification of any activatable Clostridial toxins disclosed in thepresent specification.

For immobilized metal affinity chromatography (IMAC) proteinpurification, E. coli BL21 (DE3) cell pellets used to express a modifiedClostridial toxin, as described in Example 20, will be resuspended inColumn Binding Buffer (25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM sodiumchloride; 10 mM imidazole; 2× Protease Inhibitor Cocktail Set III (EMDBiosciences-Calbiochem, San Diego Calif.); 5 units/mL of Benzonase (EMDBiosciences-Novagen, Madison, Wis.); 0.1% (v/v) TRITON-X® 100,4-octylphenol polyethoxylate; 10% (v/v) glycerol), and will then betransferred to a cold Oakridge centrifuge tube. The cell suspension willbe sonicated on ice (10-12 pulses of 10 seconds at 40% amplitude with 60seconds cooling intervals on a Branson Digital Sonifier) in order tolyse the cells and then is centrifuged (16,000 rpm at 4° C. for 20minutes) to clarify the lysate. An immobilized metal affinitychromatography column will be prepared using a 20 mL Econo-Pac columnsupport (Bio-Rad Laboratories, Hercules, Calif.) packed with 2.5-5.0 mLof TALON™ SuperFlow Co2+ affinity resin (BD Biosciences-Clontech, PaloAlto, Calif.), which will then be equilibrated by rinsing with 5 columnvolumes of deionized, distilled water, followed by 5 column volumes ofColumn Binding Buffer. The clarified lysate will be applied slowly tothe equilibrated column by gravity flow (approximately 0.25-0.3mL/minute). The column will then be washed with 5 column volumes ofColumn Wash Buffer (N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 0.1%(v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol).The modified Clostridial toxin will be eluted with 20-30 mL of ColumnElution Buffer (25 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 500 mM imidazole; 0.1%(v/v) TRITON-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol)and will be collected in approximately twelve 1 mL fractions. The amountof modified Clostridial toxin contained in each elution fraction will bedetermined by a Bradford dye assay. In this procedure, 20 μL aliquots ofeach 1.0 mL fraction will be combined with 200 μL of Bio-Rad ProteinReagent (Bio-Rad Laboratories, Hercules, Calif.), diluted 1 to 4 withdeionized, distilled water, and then the intensity of the colorimetricsignal will be measured using a spectrophotometer. The five fractionswith the strongest signal will be considered the elution peak and willbe combined together. Total protein yield will be determined byestimating the total protein concentration of the pooled peak elutionfractions using bovine gamma globulin as a standard (Bio-RadLaboratories, Hercules, Calif.).

For purification of a modified Clostridial toxin using a FPLC desaltingcolumn, a HiPrep™ 26/10 size exclusion column (Amersham Biosciences,Piscataway, N.J.) will be pre-equilibrated with 80 mL of 4° C. ColumnBuffer (50 mM sodium phosphate, pH 6.5). After the column isequilibrated, a modified Clostridial toxin sample will be applied to thesize exclusion column with an isocratic mobile phase of 4° C. ColumnBuffer and at a flow rate of 10 mL/minute using a BioLogic DuoFlowchromatography system (Bio-Rad Laboratories, Hercules, Calif.). Thedesalted modified Clostridial toxin sample will be collected as a singlefraction of approximately 7-12 mL.

For purification of a modified Clostridial toxin using a FPLC ionexchange column, a modified Clostridial toxin sample that has beendesalted following elution from an IMAC column will be applied to a 1 mLQ1™ anion exchange column (Bio-Rad Laboratories, Hercules, Calif.) usinga BioLogic DuoFlow chromatography system (Bio-Rad Laboratories,Hercules, Calif.). The sample will be applied to the column in 4° C.Column Buffer (50 mM sodium phosphate, pH 6.5) and will be eluted bylinear gradient with 4° C. Elution Buffer (50 mM sodium phosphate, 1 Msodium chloride, pH 6.5) as follows: step 1, 5.0 mL of 5% Elution Bufferat a flow rate of 1 mL/minute; step 2, 20.0 mL of 5-30% Elution Bufferat a flow rate of 1 mL/minute; step 3, 2.0 mL of 50% Elution Buffer at aflow rate of 1.0 mL/minute; step 4, 4.0 mL of 100% Elution Buffer at aflow rate of 1.0 mL/minute; and step 5, 5.0 mL of 0% Elution Buffer at aflow rate of 1.0 mL/minute. Elution of modified Clostridial toxin fromthe column will be monitored at 280, 260, and 214 nm, and peaksabsorbing above a minimum threshold (0.01 au) at 280 nm will becollected. Most of the modified Clostridial toxin will be eluted at asodium chloride concentration of approximately 100 to 200 mM. Averagetotal yields of modified Clostridial toxin will be determined by aBradford assay.

Expression of a modified Clostridial toxin will be analyzed bypolyacrylamide gel electrophoresis. Samples purified using the proceduredescribed above are added to 2×LDS Sample Buffer (Invitrogen, Inc,Carlsbad, Calif.) and will be separated by MOPS polyacrylamide gelelectrophoresis using NuPAGE® Novex 4-12% Bis-Tris precastpolyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) underdenaturing, reducing conditions. Gels will be stained with SYPRO® Ruby(Bio-Rad Laboratories, Hercules, Calif.) and the separated polypeptideswill be imaged using a Fluor-S MAX Multilmager (Bio-Rad Laboratories,Hercules, Calif.) for quantification of modified Clostridial toxinexpression levels. The size and amount of modified Clostridial toxinwill be determined by comparison to MagicMark™ protein molecular weightstandards (Invitrogen, Inc, Carlsbad, Calif.).

Expression of modified Clostridial toxin will also be analyzed byWestern blot analysis. Protein samples purified using the proceduredescribed above will be added to 2×LDS Sample Buffer (Invitrogen, Inc,Carlsbad, Calif.) and will be separated by MOPS polyacrylamide gelelectrophoresis using NuPAGE® Novex 4-12% Bis-Tris precastpolyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) underdenaturing, reducing conditions. Separated polypeptides will betransferred from the gel onto polyvinylidene fluoride (PVDF) membranes(Invitrogen, Inc, Carlsbad, Calif.) by Western blotting using aTrans-Blot® SD semi-dry electrophoretic transfer cell apparatus (Bio-RadLaboratories, Hercules, Calif.). PVDF membranes will be blocked byincubating at room temperature for 2 hours in a solution containing 25mM Tris-Buffered Saline (25 mM 2-amino-2-hydroxymethyl-1,3-propanediolhydrochloric acid (Tris-HCl) (pH 7.4), 137 mM sodium chloride, 2.7 mMpotassium chloride), 0.1% TWEEN-20®, polyoxyethylene (20) sorbitanmonolaureate, 2% bovine serum albumin, 5% nonfat dry milk. Blockedmembranes will be incubated at 4° C. for overnight in Tris-BufferedSaline TWEEN-20® (25 mM Tris-Buffered Saline, 0.1% TWEEN-20®,polyoxyethylene (20) sorbitan monolaureate) containing appropriateprimary antibodies as a probe. Primary antibody probed blots will bewashed three times for 15 minutes each time in Tris-Buffered SalineTWEEN-20®. Washed membranes will be incubated at room temperature for 2hours in Tris-Buffered Saline TWEEN-20® containing an appropriateimmunoglobulin G antibody conjugated to horseradish peroxidase as asecondary antibody. Secondary antibody-probed blots will be washed threetimes for 15 minutes each time in Tris-Buffered Saline TWEEN-20®. Signaldetection of the labeled modified Clostridial toxin will be visualizedusing the ECL Plus™ Western Blot Detection System (Amersham Biosciences,Piscataway, N.J.) and will be imaged with a Typhoon 9410 Variable ModeImager (Amersham Biosciences, Piscataway, N.J.) for quantification ofmodified Clostridial toxin expression levels.

Although aspects of the present invention have been described withreference to the disclosed embodiments, one skilled in the art willreadily appreciate that the specific examples disclosed are onlyillustrative of these aspects and in no way limit the present invention.Various modifications can be made without departing from the spirit ofthe present invention.

Those of skill in the art will understand that the Examples providedherein describe preferred compositions and methods, and that a varietyof different cloning strategies, protease cleavage sites, and specificbinding complex members may be employed in the practice and use of thepresent invention while remaining within the invention's scope.Additionally, different di-chain or binary toxin molecules and modifiedversions thereof (for example, BoNT/B-E and modified variants thereof)may be used as the basis for the methods and compositions of the presentinvention.

1. A single-chain polypeptide comprising: a) a first domain comprising abinding element comprising a melanocortin peptide able to preferentiallyinteract with a melanocortin peptide receptor under physiologicalconditions; b) a second domain comprising a translocation elementcomprising a Clostridial neurotoxin heavy chain able to facilitate thetransfer of said single-chain polypeptide across a vesicular membrane;c) a third domain comprising a therapeutic element comprising aClostridial neurotoxin light chain having biological activity whenreleased into the cytoplasm of said target cell; and d) a fourth domaincomprising an exogenous protease cleavage site;
 2. The polypeptide ofclaim 1, wherein said fourth domain intervenes between said first domainand said second domain.
 3. The polypeptide of claim 1, wherein saidfourth domain intervenes between said second domain and said thirddomain.
 4. The polypeptide of claim 1, wherein said polypeptidecomprises a linear amino-to-carboxyl single polypeptide order of 1) abinding element, a translocation element, an exogenous protease cleavagesite, and a therapeutic element, 2) a binding element, a therapeuticelement, an exogenous protease cleavage site, and a translocationelement, 3) a therapeutic element, an exogenous protease cleavage site,a binding element, and a translocation element, 4) a translocationelement, an exogenous protease cleavage site, a binding element, and atherapeutic element, 5) a therapeutic element, a binding element, anexogenous protease cleavage site, and a translocation element, 6) atranslocation element, a binding element, an exogenous protease cleavagesite, and a therapeutic element, 7) a therapeutic element, an exogenousprotease cleavage site, a translocation element, and a binding element,or 8) a translocation element, an exogenous protease cleavage site, atherapeutic element, and a binding element.
 5. The polypeptide of claim1, wherein said melanocortin peptide comprises a melanocyte stimulatinghormone, an adrenocorticotropin peptide, a lipotropin peptide, or aneuropeptide comprising SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 90.6. The polypeptide of claim 5, wherein said melanocyte stimulatinghormone comprises an α-melanocyte stimulating hormone, a β-melanocytestimulating hormone, a γ-melanocyte stimulating hormone.
 7. Thepolypeptide of claim 5, wherein said melanocyte stimulating hormonecomprises SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO:
 83. 8. Thepolypeptide of claim 5, wherein said adrenocorticotropin peptidecomprises an adrenocorticotropin or a corticotropin-like intermediarypeptide.
 9. The polypeptide of claim 5, wherein said adrenocorticotropinpeptide comprises SEQ ID NO: 84 or SEQ ID NO:
 85. 10. The polypeptide ofclaim 5, wherein said lipotropin peptide comprises β-lipotropin or aγ-lipotropin.
 11. The polypeptide of claim 5, wherein said lipotropinpeptide comprises SEQ ID NO: 86 or SEQ ID NO:
 87. 12. The polypeptide ofclaim 5, wherein said melanocortin peptide comprises a neuropeptidecomprising SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO:
 90. 13. Thepolypeptide of claim 1, wherein said translocation element comprises aClostridium botulinum neurotoxin heavy chain.
 14. The polypeptide ofclaim 13, wherein said Clostridium botulinum neurotoxin heavy chaintranslocation element is selected from the group consisting of aClostridium botulinum serotype A neurotoxin heavy chain, a Clostridiumbotulinum serotype B neurotoxin heavy chain, a Clostridium botulinumserotype C1 neurotoxin heavy chain, a Clostridium botulinum serotype Dneurotoxin heavy chain, a Clostridium botulinum serotype E neurotoxinheavy chain, a Clostridium botulinum serotype F neurotoxin heavy chainand a Clostridium botulinum serotype G neurotoxin heavy chain.
 15. Thepolypeptide of claim 1, wherein said translocation element comprises aClostridium tetani neurotoxin heavy chain.
 16. The polypeptide of claim1, wherein said therapeutic element comprises a Clostridium botulinumneurotoxin light chain.
 17. The polypeptide of claim 16, wherein saidClostridium botulinum neurotoxin light chain therapeutic element isselected from the group consisting of a Clostridium botulinum serotype Aneurotoxin light chain, a Clostridium botulinum serotype B neurotoxinlight chain, a Clostridium botulinum serotype C1 neurotoxin light chain,a Clostridium botulinum serotype D neurotoxin light chain, a Clostridiumbotulinum serotype E neurotoxin light chain, a Clostridium botulinumserotype F neurotoxin light chain and a Clostridium botulinum serotype Gneurotoxin light chain.
 18. The polypeptide of claim 1, wherein saidtherapeutic element comprises a Clostridium tetani neurotoxin lightchain.
 19. The polypeptide of claim 1, wherein said exogenous proteasecleavage site comprises a non-human enterokinase cleavage site, atobacco etch virus protease cleavage site, a tobacco vein mottling virusprotease cleavage site, a human rhinovirus 3C protease cleavage site, asubtilisin cleavage site, a hydroxylamine cleavage site, a SUMO/ULP-1protease cleavage site, or a non-human Caspase 3 protease cleavage site.20. The polypeptide of claim 19, wherein said non-human enterokinasecleavage site comprises SEQ ID NO:
 21. 21. The polypeptide of claim 19,wherein said tobacco etch virus protease cleavage site comprises SEQ IDNO: 22 or SEQ ID NO:
 23. 22. The polypeptide of claim 19, wherein saidtobacco etch virus protease cleavage site comprises SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO:
 33. 23. Thepolypeptide of claim 19, wherein said tobacco vein mottling virusprotease cleavage site comprises SEQ ID NO: 34 or SEQ ID NO:
 35. 24. Thepolypeptide of claim 19, wherein said tobacco vein mottling virusprotease cleavage site comprises SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, or SEQ ID NO:
 39. 25. The polypeptide of claim 19, wherein saidhuman rhinovirus 3C protease cleavage site comprises SEQ ID NO:
 40. 26.The polypeptide of claim 19, wherein said human rhinovirus 3C proteasecleavage site comprises SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 45 or SEQ ID NO:
 46. 27. The polypeptide of claim19, wherein said subtilisin cleavage site comprises SEQ ID NO: 47 or SEQID NO:
 48. 28. The polypeptide of claim 19, wherein said subtilisincleavage site comprises SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51.29. The polypeptide of claim 19, wherein said hydroxylamine cleavagesite comprises SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO:
 54. 30. Thepolypeptide of claim 19, wherein said non-human Caspase 3 proteasecleavage site comprises SEQ ID NO:
 57. 31. The polypeptide of claim 19,wherein said non-human Caspase 3 protease cleavage site comprises SEQ IDNO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 orSEQ ID NO:
 63. 32. The polypeptide of claim 1, wherein said polypeptidecomprises a fifth domain comprising a target-binding portion of abinding tag.
 33. A pharmaceutical composition comprising a carrier and asingle-chain polypeptide according to any one of claims 1-32
 34. Anucleotide sequence encoding a single-chain polypeptide according to anyone of claims 1-32.
 35. The nucleotide sequence of claim 34, furthercomprising an expression vector.
 36. A method of making a single-chainpolypeptide comprising: a) inserting a nucleotide sequence of claims 35into a suitable host cell; b) growing said host cell in culture; and c)permitting or inducing the host cell to express the single chainpolypeptide encoded by said nucleotide sequence.
 37. A method ofpurifying a single chain-polypeptide comprising: a) lysing a host cellcontaining a nucleotide sequence expressing a single-chain polypeptideto produce a cell lysate, said single-chain polypeptide according to anyone of claims 1-32; b) contacting said cell lysate with a targetcompound so as to form a specific binding complex capable of beingimmobilized comprising said binding tag and said target compound; and c)separating said binding complex from said cell lysate.
 38. A method ofactivating a single-chain polypeptide, the method comprising the step ofincubating an single-chain polypeptide according to any one of claims1-32 with an exogenous protease; wherein the exogenous protease cleavesthe exogenous protease cleavage site; and wherein cleavage of thesingle-chain polypeptide by the exogenous protease converts thesingle-chain polypeptide from its single-chain polypeptide form into itsdi-chain form, thereby activating the single-chain polypeptide.
 39. Apharmaceutical composition comprising a carrier and a single-chainpolypeptide activated according to claim 38.